Patent Application
20170107213
The invention relates to the medical field. Specifically, the invention relates to substituted nitrogen-containing heterocyclic derivatives used as Akt inhibitors, pharmaceutical compositions comprising the same and applications of antitumor thereof.
Therioma is one of the major diseases threatening human health. During 2008, there are about 12.7 million new cancer patients and 7.6 million patients died from cancer worldwide. It is estimated that by 2020, new cancer patients worldwide will increase to 15 million, and the death toll caused by cancer are also rising rapidly all over the world, which may increase to 13.2 million. The prevention and treatment of tumors has become an important research topic in the medical field among countries. Although there have been some antitumor drugs in clinical use, in general, the toxic, serve side effects and drug resistance of these drugs are often observed in clinic, which greatly limit the clinical treatment. Therefore, it is of great significance to develop novel antitumor drugs with high efficiency and low toxicity.
Suppression of cell apoptosis is closely related to the occurrence and development of tumors, which is believed to be one of the important reasons for drug resistance of cancer cells. The study of apoptosis signaling pathway provides a new idea for antitumor therapy, and also provides a new target for developing novel antitumor drugs. In 1995, Akt, also known as protein kinase B, was found as the downstream target of PI3K activated by various growth factors. Akt is at the core position in the PI3K/Akt signaling pathway, and Akt family members have three subtypes including Akt1, Akt2 and Akt3, with more than 80% sequence identity. It is found in the study that the different subtypes of Akt are highly consistent in view of structure and function, except for the expression levels in different tumors. Akt can directly phosphorylate mTOR, Bad and Caspase 9 protein, as well as control Fork Head transcription factor family and NF-κB for further controlling the significant cell biological process in the occurrence and development of tumors, such as transcription, translation, metabolism, apoptosis, angiogenesis, and so on. It is also found in the study that the phenomenon of Aid overexpression or activity disorders exists in most of tumors, and Akt abnormity is closely related to the occurrence and development of these tumors as well as the generated resistance to chemotherapy and radiotherapy. It has been proved that by vivo and in vitro pharmacological experiments that Akt inhibitors can promote programed death of cancer cells. Therefore, Akt has attracted increasing attention as a potential antitumor target.
Akt inhibitors being in clinical research can be classified into: ATP-competitive inhibitors such as AZD5363, GSK-2110183 (afuresertib), GDC-0068 (Ipatasertib); allosteric inhibitors such as MK-2206; pH-domain binding inhibitors such as Perifosine. GSK-2110183 is an oral Akt inhibitor developed by GSK, and the single-drug thereof exhibits good safety and clinical activity against hematologic malignancies (including multiple myeloma), which is currently in phase II clinical trials. GDC-0068 is a highly selective pan-Akt inhibitor, the drug combination thereof with docetaxel or mFOLFOX-6 is well-tolerated in patients suffered from advanced solid tumors and exhibits preliminary signs of anti-tumor activity, and the dose-escalation trial thereof is still in clinical phase II. MK2206 is a 2,3-diphenyl quinoxalines derivative, which is currently in clinical phase II for the treatment of many kinds of cancer, such as gastric cancer, breast cancer etc. The clinical datas of above compounds confirm that the treatment strategy has good selectivity and improved tumor sensibility to chemotherapy and radiotherapy. Therefore, the development of novel Akt inhibitors is expected to provide clinical medicine with new therapeutic mechanism for tumor therapy.
The object of the invention is to provide a novel substituted nitrogen-containing heterocyclic derivatives, an optical isomers thereof, or pharmaceutically acceptable salts or solvate thereof, with strong antitumor effect and Akt inhibition.
Terminology Note: The term “aryl”, as used herein, refers to a group of an all-carbon single ring or a polycyclic fused ring containing 5 to 12 carbon atoms with fully conjugated it electron system. Non-limiting examples of the aromatic ring include benzene ring, naphthalene ring and anthracene ring. The aromatic ring may be unsubstituted or substituted. Substituents of the aromatic ring are selected from the group consisting of halogen, nitro, amino, cyano, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, halogenated C1-C6 alkoxy, C3-C6 cycloalkyl, halogenated C3-C6 cycloalkyl.
The term “heterocyclic aryl”, as used herein, refers to a group of unsaturated carbon ring containing 5 to 12 annular atoms, wherein one or more carbon atoms are replaced by heteroatom such as oxygen, nitrogen or sulfur. The heterocyclic aromatic ring can be a single ring or a dual ring fused by two rings. Special heterocyclic aryl may be: pyridinyl, pyrimidinyl, pyrazinyl, isoxazolyl, isothiazolyl, pyrazolyl, thiazolyl, oxazolyl, imidazolyl, and the like. The heterocyclic aryl may be unsubstituted or substituted. Substituents of the heterocyclic aryl are selected from the group consisting of halogen, nitro, amino, cyano, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, halogenated C1-C6 alkoxy, C3-C6 cycloalkyl, halogenated C3-C6 cycloalkyl.
The term “heterocyclic alkyl”, as used herein, refers to a group of a single ring or a polycyclic fused ring with 5 to 9 annular atoms in its ring, wherein one or two annular atoms is heteroatom selected from the group consisting of N, O or S(O)m (wherein m is an integer from 0 to 2) and other annular atoms are carbon atoms. These rings may have one or more double bonds, but does not have fully conjugated π electron system. The unsubstituted heterocyclic alkyl may be pyrrolidinyl, piperidyl, piperazinyl, morpholinyl, thiomorpholinyl, homopiperazinyl, and the like. The heterocyclic ring may be unsubstituted or substituted. Substituents of the heterocyclic ring are selected from the group consisting of halogen, nitro, amino, cyano, hydroxyl, C1-C6 alkyl, C1-C6 alkoxy, halogenated C1-C6 alkyl, halogenated C1-C6 alkoxy, C3-C6 cycloalkyl, halogenated C3-C6 cycloalkyl.
The term “cycloalkyl”, as used herein, refers to a group of a saturated single carbon ring with 3 to 6 carbon atoms, unless different number of atoms is specified. “Cycloalkyl” includes, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl and cyclooctyl. “Cycloalkyl” also includes substituted cycloalkyl. The cycloalkyl may optionally be substituted on any available carbon with one or more substituents selected from the group consisting of alkoxy, halogen, and haloalkyl (e.g., perfluoroalkyl).
The term “alkoxy”, as used herein, refers to the —O-alkyl group, wherein alkyl is defined as above. Examples of “alkoxy” as used herein include, but are not limited to, methoxyl, ethoxyl, n-propoxyl, isopropoxyl, n-butoxyl and t-butoxyl. “Alkoxy” also includes substituted alkoxy. The alkoxy groups may be optionally substituted with halogen for one or more times.
The term “halogen”, as used herein, refers to fluorine, chlorine, bromine or iodine, preferably, fluorine or chlorine.
The term “pharmaceutically acceptable derivatives” refers to salts and solvates of the selected compound.
The term “solvate”, as used herein, refers to a complex of variable stoichiometry formed by solute (e.g., compounds of formulas (I) to (XII) of this invention) and solvent. In view of the purpose of the invention, solvents do not interfere the biological activity of the solute. Examples of suitable solvent include, but are not limited to, water, methanol, ethanol and acetic acid. Preferably the solvent used is a pharmaceutically acceptable solvent. Examples of suitable pharmaceutically acceptable solvent include, but are not limited to, water, ethanol and acetic acid. More preferably, the solvent used is water.
This invention adopts the technical schemes as follows:
This invention provides substituted nitrogen-containing heterocyclic derivatives with the structure of general formula (I):
and optical isomers thereof, or pharmaceutically acceptable salts or solvate thereof, wherein:
Ring A is selected from the group consisting of unsubstituted or substituted 5- or 6-membered aryl, 5- or 6-membered heterocyclic aryl containing 1 to 4 heteroatoms selected from O, N and S; that is, ring A may be a unsubstituted or substituted 5-membered aryl; ring A may also be a unsubstituted or substituted 6-membered aryl.
B is selected from the group consisting of
wherein B1 and B2 are same or different; B1 and B2 are each independently selected from the group consisting of O, N(Ra), C(Rb)(Rc) or are absent, wherein Ra, Rb and Rc are each independently selected from the group consisting of H, C1-C4 alkyl, halogenated C1-C4 alkyl, C1-C4 alkoxy, halogenated C1-C4 alkoxy;
Ring C is selected from the group consisting of 5-8 membered saturated or unsaturated aliphatic nitrogen-containing heterocyclic ring which is unsubstituted or substituted; that is, ring C may be a 5-8 membered saturated aliphatic nitrogen-containing heterocyclic ring without substitution, a 5-8 membered saturated aliphatic nitrogen-containing heterocyclic ring which is substituted, a 5-8 membered unsaturated fatty nitrogen-containing heterocyclic ring without substitution, a 5-8 membered unsaturated aliphatic nitrogen-containing heterocyclic ring which is substituted; preferably, ring C is the following aliphatic nitrogen-containing heterocyclic ring:
R1 is selected from the group consisting of H, C1-C4 alkyl, halogenated C1-C4 alkyl,
wherein n is an integer from 0 to 4, Rd is selected from the group consisting of H, C1-C4 alkyl, halogenated C1-C4 alkyl, Re is selected from the group consisting of C1-C4 alkyl, halogenated C1-C4 alkyl, C1-C4 alkoxy, halogenated C1-C4 alkoxy;
R2 is selected from the group consisting of unsubstituted or substituted aryl, unsubstituted or substituted heterocyclic aryl, unsubstituted or substituted cycloalkyl, unsubstituted or substituted saturated heterocyclic alkyl, unsubstituted or substituted unsaturated heterocyclic alkyl, aryl and heterocyclic aryl which is optionally fused;
m is an integer from 0 to 3;
R3 is selected from the group consisting of amino, cyano, C1-C4 alkyl, halogenated C1-C4 alkyl, C1-C4 alkoxy, C1-C4 carboxyl, halogenated C1-C4 alkoxy,
wherein n is an integer from 0 to 4, Rd is selected from the group consisting of H, C1-C4 alkyl, halogenated C1-C4 alkyl, C1-C4 alkoxy, halogenated C1-C4 alkoxy, Re is selected from the group consisting of C1-C4 alkyl, halogenated C1-C4 alkyl, C1-C4 alkoxy, halogenated C1-C4 alkoxy; ring D is selected from saturated or unsaturated 5-8 membered aliphatic nitrogen-containing heterocyclic ring; that is, ring D may be a 5-8 membered saturated aliphatic nitrogen-containing heterocyclic ring without substitution, a substituted 5-8 membered saturated aliphatic nitrogen-containing heterocyclic ring, a 5-8 membered unsaturated aliphatic nitrogen-containing heterocyclic ring without substitution, a substituted 5-8 membered unsaturated aliphatic nitrogen-containing heterocyclic ring; preferably, ring D is the following aliphatic nitrogen-containing heterocyclic ring:
R4, R5 are each independently selected from the group consisting of H, halogen, nitro, amino, cyano, hydroxyl, C1-C4 alkyl, halogenated C1-C4 alkyl, C1-C4 alkoxy, halogenated C1-C4 alkoxy, unsubstituted or substituted furanyl, thiophenyl, phenyl, pyridinyl;
R6 is selected from the group consisting of H, C1-C4 alkyl, halogenated C1-C4 alkyl, C1-C4 alkoxy, halogenated C1-C4 alkoxy;
Q, Y are each independently selected from N and —C(Rf)—; Z is selected from N and —C(Rg)—; at least one group of Q, Y and Z is N atom and at most two groups are the same; wherein Rf is selected from H, halogen; Rg is selected from the group consisting of H, halogen, hydroxyl, carboxyl, hydroxymethyl, saturated or unsaturated C1-C4 alkyl, halogenated C1-C4 alkyl, C1-C4 alkoxy, halogenated C1-C4 alkoxy, unsubstituted or substituted aryl, unsubstituted or substituted heterocyclic aryl, unsubstituted saturated or partly saturated heterocyclic ring, substituted saturated or partly saturated heterocyclic ring, unsubstituted or substituted cycloalkyl;
said substituent is optionally selected from the group consisting of halogen, nitro, amino, cyano, hydroxyl, C1-C4 alkyl, halogenated C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkylamino, halogenated C1-C4 alkoxy, halogenated C1-C4 alkylamino.
Further, the preferred compounds in the invention have a structure of general formula (II):
and optical isomers thereof, or pharmaceutically acceptable salts or solvate thereof, wherein: Ring A, B, R1, m, R2, R3, R4, R5, Q, Y, Z and R6 are as defined in the general formula (I);
V is selected from (CH2)m1, wherein m1 is an integer from 0 to 3.
Particularly, the preferred compounds in the invention have the structure of general formula (III):
and optical isomers, or pharmaceutically acceptable salts or solvates thereof, wherein:
R1, R2, R3, R4, R5, Q, Y, Z, B, m and R6 are as defined in the general formula (I);
X is selected from the group consisting of O, S, N(Rh), wherein Rh is selected from the group consisting of H, C1-C5 alkyl, halogenated C1-C5 alkyl, C1-C5 alkoxy and halogenated C1-C5 alkoxy; L is selected from the group consisting of CH and N.
Particularly, the preferred compounds in the invention have the structure of general formula (IV):
and optical isomers, or pharmaceutically acceptable salts or solvates thereof, wherein:
R2, L are as defined in the general formula (III); R4, R5 are each independently selected from halogen and C1-C3 alkyl;
X is selected from the group consisting of O, S, NH and NCH3; Z is selected from —C(Rg)—, Rg is preferably selected from the group consisting of H, halogen and C1-C3 alkyl.
In addition, the preferred compounds in the invention have the structure of general formula (V):
and optical isomers, or pharmaceutically acceptable salts or solvates thereof, wherein:
R1, R2, R3, B, m, Q, Y, Z, R6 are as defined in the general formula (I);
V is selected from (CH2)m1, wherein m1 is an integer from 0 to 3;
E and T are same or different, E and T are each independently selected from N and —C(Ri)—, wherein Ri is selected from the group consisting of H, halogen, cyano, nitro, amino, C1-C4 alkyl, halogenated C1-C4 alkyl, C1-C4 alkoxy, halogenated C1-C4 alkoxy, unsubstituted or substituted furanyl, unsubstituted or substituted thiophenyl, unsubstituted or substituted phenyl, unsubstituted or substituted pyridinyl.
Further, the preferred compounds in the invention have the structure of general formula (VI):
and optical isomers, or pharmaceutically acceptable salts or solvates thereof, wherein:
E, T, R1, R2, R3, B, Q, Y, Z, m and R6 are as defined in the general formula (V);
More particularly, the preferred compounds in the invention have the structure of general formula (VII):
and optical isomers, or pharmaceutically acceptable salts or solvates thereof, wherein:
R2 is as defined in the general formula (I); E, T are each independently selected from N and —C(Ri)—, wherein Ri is selected from the group consisting of H, halogen and C1-C3 alkyl; Z is selected from —C(Rg)—, wherein Rg is selected from the group consisting of H, halogen, C1-C3 alkyl.
In addition, the preferred compounds in the invention have the structure of general formula (VIII):
and optical isomers, or pharmaceutically acceptable salts or solvates thereof, wherein:
Ring A, Rg, R2, R3, R4, R5 are as defined in the general formula (I).
Further, the preferred compounds in the invention have the structure of general formula (IX):
and optical isomers, or pharmaceutically acceptable salts or solvates thereof, wherein:
Rg, R3 and R4 are as defined in the general formula (I);
R2 is preferably selected from unsubstituted or substituted aryl, unsubstituted or substituted heterocyclic aryl; said substituent is optionally selected from the group consisting of halogen, nitro, amino, cyano, hydroxyl, C1-C4 alkyl, halogenated C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkylamino, halogenated C1-C4 alkoxy, halogenated C1-C4 alkylamino;
E, T are each independently selected from N and —C(Ri)—, wherein Ri is selected from the group consisting of H, halogen, C1-C3 alkyl, halogenated C1-C3 alkyl.
Still further, the preferred compounds in the invention have the structure of general formula (X):
and optical isomers, or pharmaceutically acceptable salts or solvates thereof, wherein:
Rg, R2, R3 and R4 are as defined in the general formula (I); X is selected from the group consisting of O, S, NH and NCH3; L is selected from CH and N.
Further, the preferred compounds in the invention have the structure of general formula (XI):
and optical isomers, or pharmaceutically acceptable salts or solvates thereof, wherein:
Rg, R2, R3 and R4 are as defined in the general formula (I); X is selected from O, S.
Finally, the preferred compounds in the invention have the structure of general formula (XII):
and optical isomers, or pharmaceutically acceptable salts or solvates thereof, wherein:
Rg, R2, R3 and R4 are as defined in the general formula (I).
Specifically, according to the general formula (IV), the preferred compounds in the invention are:
Specifically, according to the general formula (VII), the preferred compounds in the invention are:
Specifically, according to the general formula (IX), the preferred compounds in the invention are:
Specifically, according to the general formula (XI), the preferred compounds in the invention are:
Specifically, according to the general formula (XII), the preferred compounds in the invention are:
The invention adopts the methods well-known to one skilled in the art for preparing the salts of the substituted nitrogen-containing heterocyclic compounds. Said salts may be salts of organic acid, salts of inorganic acid, and the like. Said salts of organic acid comprise citrate, fumarate, oxalate, malate, lactate, camphorsulfonate, p-toluenesulfonate, mesylate, and the like; said salts of inorganic acid comprise hydrohaloride, sulfate, phosphate, nitrate, and the like. For example, mesylate or trifluoromethanesulfonate may be formed with a lower alkyl sulfonic acid, such as methanesulfonic acid, trifluoromethanesulfonic acid, and so on; p-toluenesulfonate or benzene sulfonate may be formed with an aryl sulphonic acid, such as benzenesulfonic acid, p-toluenesulfonic acid, and so on; the corresponding salts may be formed with an organic carboxylic acid, such as acetic acid, fumaric acid, tartaric acid, oxalic acid, maleic acid, malic acid, succinic acid or citric acid and the like; glutamate or aspartate may be formed with an amino acid, such as glutamic acid or aspartic acid, and so on. The corresponding salts may be formed with an inorganic acid, such as haloid acid (such as hydrofluoric acid, hydrobromic acid, hydroiodic acid, hydrochloric acid), nitric acid, carbonic acid, sulfuric acid, or phosphoric acid, and the like.
The second object of the invention is to provide a pharmaceutical composition, comprising at least one active component and one or more pharmaceutically acceptable carriers or excipients, wherein said one active component may be any one or more selected from the group consisting of the substituted nitrogen-containing heterocyclic compounds with the structure of general formula (I) to (XII) in the invention and preferred compounds thereof, optical isomers of said compounds, pharmaceutically acceptable salts of the said compounds or the optical isomers thereof, solvates of the said compounds or the optical isomers thereof.
The carriers comprise conventional diluents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorption carries, lubricants, and the like in the pharmaceutical field, if needed, odorants, edulcorants and the like may also be added. The pharmaceutical composition in the present invention may be made into various forms such as tablets, powders, granules, capsules, oral liquid and drug for injection, and the like, and all the above dosage form can be prepared according to the conventional methods in the pharmaceutical field.
The invention also provides the use of compounds of general formula (I) to (XII) and optical isomers thereof, or pharmaceutically acceptable salts or solvates thereof for preparing antitumor drugs. Said tumor is breast cancer, sarcoma, lung cancer, prostate cancer, colon cancer, rectal cancer, kidney cancer, pancreatic cancer, leukemia, neuroblastoma, glioma, head cancer, neck cancer, thyroid cancer, liver cancer, ovarian cancer, vulvar cancer, cervical cancer, endometrial cancer, testicular cancer, bladder cancer, esophageal cancer, gastric cancer, nasopharyngeal carcinoma, buccal cancer, oral cancer, gastrointestinal stromal tumor, skin cancer and multiple myeloma.
Each compound of general formula (I) may be conveniently prepared by separately preparing three constituents of said compound, followed by synthesizing the compound of the general formula (I) with those constituents. For convenience, said three constituents are referred to herein as the head, the core, and the tail. When used individually, the head, core, and tail used throughout refer to each constituent, and also refer to the corresponding moiety when present in form of combination of head/core, tail/core, and/or head/core/tail hereins.
The head component of the compounds of general formula (I) in the present invention is a substituted nitrogen-containing heterocyclic borate or boronic acid compound represented by head (XIII); the core component is a compound with the structure of 5- or 6-membered aryl sulfonyl chloride or aryl formic acid or arylamine substituted with bromine or iodine or the like represented by the core of general formula (XIV), the tail component is a compound with the structure of substituted 5-8 membered saturated or unsaturated aliphatic nitrogen-containing heterocyclic formic acid or nitrogen-containing amino heterocyclic ring or the like represented by the tail of general formula (XV), as shown below:
The head (XIII), core (XIV), and tail (XV) of the compounds form the general formula (I) by the synthetic route as shown below:
Specifically, the compounds of the general formula (VIII) in the present invention may be obtained by constituted with the synthetic route as shown below, by using N-methyl pyrazole borate or boronic acid compound represented by the head (XIII-1), 5- or 6-membered aryl formic acid compound substituted by bromine or iodine represented by the core (XIV-1), piperidine compound substituted by protecting groups represented by the tail (XV).
Wherein, PG is a common amino protective group, such as: Boc (t-butyloxycarboryl), Cbz (carboxybenzyl), Ac (acetyl) and the like.
More specifically, it is illustrated as represented by the compounds of the general formula (IX) and (X).
The head component of the general formula (IX) and (X) in the present invention is a pyrazol boric acid pinacol ester compound represented by the head (i):
Wherein Rg is as defined in the general formula (I), i.e., Rg is selected from the group consisting of H, halogen, hydroxyl, carboxyl, hydroxymethyl, saturated or unsaturated C1-C4 alkyl, halogenated C1-C4 alkyl, C1-C4 alkoxy, halogenated C1-C4 alkoxy, unsubstituted or substituted aryl, unsubstituted or substituted heterocyclic aryl, saturated or partly saturated heterocyclic ring which is unsubstituted or substituted, unsubstituted or substituted cycloalkyl.
The core component of the general formula (IX) and (X) in the present invention is a 6- or 5-membered aryl formic acid compound substituted by bromine represented by the core of the general formula (ii-1) and (ii-2):
Wherein E, T are as defined in the general formula (IX), i.e., E, T are each independently selected from N and —C(Ri)—, wherein Ri is selected from the group consisting of H, halogen, C1-C3 alkyl or halogenated C1-C3 alkyl;
Wherein L, X are as defined in the general formula (X), i.e., X is selected from the group consisting of O, S, NH and NCH3; L is selected from CH and N;
The substituent R4 is as defined in the general formula (I), i.e., R4 is selected from the group consisting of H, halogen, nitro, amino, cyano, C1-C4 alkyl, halogenated C1-C4 alkyl, C1-C4 alkoxy, halogenated C1-C4 alkoxy, unsubstituted or substituted furan, thiophene, phenyl, pyridinyl;
The tail component of the compounds in the present invention is a substituted piperidine compound protected by a Boc, which is represented by the tail of the general formula (iii):
wherein the substituent R2, R3 are as defined in the general formula (I), i.e., R2 is selected from the group consisting of unsubstituted or substituted aryl, unsubstituted or substituted heterocyclic aryl, unsubstituted or substituted cycloalkyl, saturated or unsaturated heterocyclic alkyl which is unsubstituted or substituted, aryl which is optionally fused, heterocyclic aryl; R3 is selected from the group consisting of amino, cyano, C1-C4 alkyl, halogenated C1-C4 alkyl, C1-C4 alkoxy, C1-C4 carboxyl, halogenated C1-C4 alkoxy,
wherein n is an integer from 0 to 4, Rd is selected from the group consisting of H, C1-C4 alkyl, halogenated C1-C4 alkyl, C1-C4 alkoxy, halogenated C1-C4 alkoxy, Re is selected from the group consisting of C1-C4 alkyl, halogenated C1-C4 alkyl, C1-C4 alkoxy, halogenated C1-C4 alkoxy, ring D is selected from 5-8 membered saturated or unsaturated aliphatic nitrogen-containing heterocyclic ring which is unsubstituted or substituted; said substituent is selected from the group consisting of halogen, nitro, amino, cyano, hydroxyl, C1-C3 alkyl, halogenated C1-C3 alkyl, C1-C3 alkoxy, halogenated C1-C3 alkoxy.
Said substituent is selected from the group consisting of halogen, nitro, amino, cyano, hydroxyl, C1-C4 alkyl, halogenated C1-C4 alkyl, C1-C4 alkoxy, C1-C4 alkylamino, halogenated C1-C4 alkoxy, halogenated C1-C4 alkylamino.
The head (i), core (ii) and tail (iii) of the general formula of the compounds may be combined by the synthetic route as shown below:
As shown in the figure, head (i) and a core (ii) of the general formula conduct Suzuki coupling reaction at heat under the condition of catalyzing by a zero valent palladium catalyst, and the obtained product is reacted with a tail (iii) under the action of condensation agent (such as EDCI), followed by that the Boc protective group of obtained product is deprotected in an acidic condition, thus obtained the compounds of the general formula (IX) and (X).
Other compounds of general formula may be prepared and obtained by referring to the preparation method of the compounds of the general formula (IX) and (X).
The present invention also provides the use of said compounds of the present invention or pharmaceutically acceptable salts thereof in the preparation of Akt inhibitors, in particular, in the preparation of medicine for treating cell proliferative diseases. Said cell proliferative diseases comprise cancer. In other words, the present invention provides the use of the substituted nitrogen-containing heterocyclic compounds or pharmaceutically acceptable salts thereof which is used alone or in combination with other drugs in the treatment of proliferative diseases (such as cancer). The antitumor drugs which may be used in combination with the compounds provided in the present invention or pharmaceutically acceptable salts thereof comprise, but are not limited to, at least one of the following group consisting of: mitotic inhibitors (such as vinblastine, vindesine, and vinorelbine); tubulin depolymerization inhibitors (such as Taxol); alkylating reagent (such as cisplatin, carboplatin and cyclophosphamide); antimetabolite (such as 5-fluorouracil, tegafur, methotrexate, cytarabine and hydroxycarbamide); insertable antibiotics (such as doxorubicin, mitomycin and bleomycin); enzymes (such as asparaginase); topoisomerase inhibitors (such as etoposide and camptothecin); biological response modifier (such as interferon); proteasome inhibitors (such asbortezomib).
It is identified by inventors of the present invention through several experiments that: the compounds of the present invention have significant inhibitory effects on Akt1, showing potent antiproliferative effects to tumor cell strains such as human ovarian cancer cell strains (OVCAR-8), colon cancer cell strains (HCT116) and the like. Therefore, the compounds of the present invention may be applied as AKT inhibitors in the drugs for treating solid tumors or leukemia in human and animals which is associated with cell proliferation.
Hereinafter, the following examples are presented to illustrate the feasibility of the present invention, and it should be understood for one skilled in the art that, modifications or alternatives of the corresponding technical features according to the teaching of the prior art still be included within the scope sought to protect in the present invention.
Sequentially adding 4-bromo furan-2-methyl formate (Compound 1-1) (4.7 g, 22.9 mmol), tetra(triphenylphosphine)palladium (0.582 g, 1.145 mmol), 1-methyl-1H-pyrazol-5-boric acid pinacol ester (5.25 g, 25.2 mmol) and potassium carbonate (7.9 g, 57.25 mmol) into a 100 mL double-neck flask under the protection of N2, adding 1,4-dioxane (30 mL) and water (6 mL) thereto, and reacting at 90V for 12 h. After the reaction is finished, the product is cooled to room temperature, extracting the reaction liquid with ethyl acetate for 3 times, washing the merged organic phase with saturated sodium chloride once, drying it with anhydrous sodium sulfate. Recycling the solvent under reduced pressure, and carrying out column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=4:1, increasing the polarity to 1:1), 3.75 g of light yellow solid (Intermediate 1-2) is obtained and the yield is 75%.
Dissolving Intermediate 1-2 (1.87 g, 6.8 mmol) in methanol (10 ml), and slowly adding 11.3 ml of 6N NaOH aqueous solution at room temperature, reacting at room temperature for 12 h, monitor the reaction with TLC thin-layer chromatography for whether it is completed, and recycling the solvent under reduced pressure after the reaction. Adding 10 ml of water to the remained reaction mixture, neutralizing NaOH in the reaction liquid with 1N HCl to pH of about 3, extracting the reaction liquid with ethyl acetate for 3 times, washing the merged organic phase with saturated sodium chloride once, and drying it with anhydrous sodium sulfate. Recycling the solvent under reduced pressure, directly obtaining 1.58 g of light yellow solid (Intermediate 1) and the yield is 89%.
Dissolving 4-(1-methyl-1H-pyrazol-5-yl)furan-2-methyl formate (Intermediate 1-2) (6.18 g, 30 mmol), N-chlorosuccinimide (8.01 g, 60 mmol) in the mixed solution of tetrahydrofuran (30 ml) and N,N-dimethylformamide (5 ml), raising temperature to 100° C., reacting for 5 h under seal. Monitoring the reaction with TLC thin-layer chromatography for whether it is completed, cooling the product to room temperature after the reaction is finished, recycling the solvent under reduced pressure, washing the remaining mixture with saturated NaHCO3 aqueous solution, and extracting the reaction liquid with ethyl acetate for 3 times, washing the merged organic phase with saturated sodium chloride once, and drying it with anhydrous sodium sulfate. Recycling the solvent under reduced pressure, carrying out column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=4:1), 6.18 g of light yellow solid (Intermediate 1-3) is obtained and the yield is 75%.
Dissolving 4-(1-methyl-1H-pyrazol-5-yl)furan-2-methyl formate (Intermediate 1-2) (6.18 g, 30 mmol), N-chlorosuccinimide (4.0 g, 30 mmol) in tetrahydrofuran (30 ml), raising temperature to 70° C., reacting for about 2 h. Monitoring the reaction with TLC thin-layer chromatography for whether it is completed, cooling the product to room temperature after the reaction is finished, recycling the solvent under reduced pressure, washing the remaining mixture with saturated NaHCO3 solution, and extracting the reaction liquid with ethyl acetate for 3 times, washing the merged organic phase with saturated sodium chloride once, and drying it with anhydrous sodium sulfate. Recycling the solvent under reduced pressure, carrying out column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=4:1), 3.60 g of light yellow solid (Intermediate 1-4) is obtained and the yield is 50%.
The synthesis steps refer to step 2 of Example 1. Intermediate 2 (2.3 g, yield of 88%) and Intermediate 3 (2.1 g, yield of 87%) are prepared from 5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-methyl formate (Intermediate 1-3) (2.75 g, 10.0 mmol) and 4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-methyl formate (Intermediate 1-4) (2.40 g, 10.0 mmol) by the similar synthesis methods as that of Intermediate 1, respectively.
Dissolving 4-(1-methyl-1H-pyrazol-5-yl)furan-2-methyl formate (Intermediate 1-2) (6.18 g, 30 mmol), N-chlorosuccinimide (5.34 g, 30 mmol) in tetrahydrofuran (40 ml), raising temperature to 65° C., reacting for about 2 h. Monitoring the reaction with TLC thin-layer chromatography for whether it is completed, cooling the product to room temperature after the reaction is finished, recycling the solvent under reduced pressure, washing the remaining mixture with saturated NaHCO3 solution, extracting the reaction liquid with ethyl acetate for 3 times, washing the merged organic phase with saturated sodium chloride once, and drying it with anhydrous sodium sulfate. Recycling the solvent under reduced pressure, carrying out column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=4:1), 10.60 g of light yellow solid (Intermediate 1-5) is obtained and the yield is 62%.
The synthesis steps refer to Step 2 of Example 1. Intermediate 4 (2.4 g, yield of 89%) is prepared from 4-(4-bromo-1-methyl-1H-pyrazol-5-yl)furan-2-methyl formate (Intermediate 1-5) (2.85 g, 10.0 mmol) by the similar synthesis method as that of Intermediate 1.
The synthesis steps refer to Step 1 of Example 1. Intermediate 1-7 (1.3 g, yield of 67%) is prepared from 4-bromothiophene formaldehyde (Compound 1-6) (1.91 g, 10.0 mmol) by the similar synthesis method as that of Intermediate 1-2.
Dissolving 4-(1-methyl-1H-pyrazol-5-yl)thiophene-2-formaldehyde (Intermediate 1-7) (0.24 g, 1.25 mmol) in methanol (5 ml), then slowly adding the aqueous solution (5 ml) dissolved with KMnO4 (0.196 g, 1.25 mmol) and Na2HPO4 (0.178 g, 1.25 mmol), stirring for 2 h at room temperature. Monitoring the reaction with TLC thin-layer chromatography for whether it is completed, after the reaction, adding 1N HCl solution dissolved with Na2SO3 (0.2 g) and saturated NaCl into reaction liquid. Vacuum filtrating the above mixed solution, extracting the reaction liquid with ethyl acetate for 3 times, washing the merged organic phase with saturated sodium chloride once, and drying it with anhydrous sodium sulfate. Recycling the solvent under reduced pressure, 0.20 g of white solid (Intermediate 5) is obtained and the yield is 77%.
Dissolving 4-(1-methyl-1H-pyrazol-5-yl)thiophene-2-formaldehyde (Intermediate 1-7) (191 mg, 1 mmol), N-chlorosuccinimide (266 mg, 2 mmol) in tetrahydrofuran (10 ml), raising temperature to 80° C. and reacting for about 2 h. Monitoring the reaction with TLC thin-layer chromatography for whether it is completed, cooling the product to room temperature after the reaction, recycling tetrahydrofuran under reduced pressure, then extracting the reaction liquid with ethyl acetate for 3 times, washing the merged organic phase with saturated sodium chloride once, and drying it with anhydrous sodium sulfate. Recycling the solvent under reduced pressure, carrying out column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=4:1), 130 mg of light yellow solid (Intermediate 1-8) is obtained and the yield is 58%.
The synthesis steps refer to Step 1 of Example 3. Intermediate 6 (1.9 g, yield of 78%) is prepared from 4-(4-chloro-1-methyl-1H-pyrazol-5-yl)thiophene-2-formaldehyde (Intermediate 1-8) (2.26 g, 10.0 mmol) with the similar synthesis method as that of Intermediate 5.
The synthesis steps refer to Step 1 of Example 3. Intermediate 1-9 (2.5 g, yield of 92%) is prepared from 4-(1-methyl-1H-pyrazol-5-yl)thiophene-2-formaldehyde (Intermediate 1-7) (1.92 g, 10.0 mmol) with the similar synthesis method as that of Compound 1-5.
The synthesis steps refer to Step 2 of Example 4. Intermediate 7 (2.6 g, yield of 91%) is prepared from 4-(4-bromo-1-methyl-1H-pyrazol-5-yl)thiophene-2-formaldehyde (Intermediate 1-9) (2.71 g, 10.0 mmol) with the similar synthesis method as that of Intermediate 5.
The synthesis steps refer to Step 1 of Example 1. Intermediate 1-11 (1.8 g, yield of 88%) is prepared from 5-methyl-4-bromothiophene-2-formaldehyde (Compound 1-10) (2.05 g, 10.0 mmol) with the similar synthesis method as that of Intermediate 1-2.
The synthesis steps refer to Step 1 of Example 5. Intermediate 1-12 (2.0 g, yield of 83%) is prepared from 5-methyl-4-(1-methyl-1H-pyrazol-5-yl)thiophene-2-formaldehyde (Intermediate 1-11) (2.06 g, 10.0 mmol) with the similar synthesis method as that of Intermediate 1-8.
The synthesis steps refer to Step 2 of Example 4. Intermediate 8 (2.13 g, yield of 83%) is prepared from 5-methyl-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)thiophene-2-formaldehyde (Intermediate 1-12) (2.41 g, 10.0 mmol) with the similar synthesis method as that of Intermediate 5.
The synthesis steps refer to Step 1 of Example 1. Intermediate 1-14 (2.12 g, yield of 81%) is prepared from 5-ethyl-4-bromothiophene-2-formaldehyde (Compound 1-13) (2.19 g, 10.0 mmol) with the similar synthesis method as that of Intermediate 1-2.
The synthesis steps refer to Step 2 in Example 4. Intermediate 9 (2.19 g, yield of 79%) is prepared from 5-ethyl-4-(4-isopropyl-1-methyl-1H-pyrazol-5-yl)thiophene-2-formaldehyde (Intermediate 1-14) (2.6 g, 10.0 mmol) with the similar synthesis method as that of Intermediate 5.
The synthesis steps refer to Step 1 in Example 1. Intermediate 1-16 (1.72 g, yield of 83%) is prepared from 5-methyl-4-bromothiazole-2-formaldehyde (Compound 1-15) (2.06 g, 10.0 mmol) with the similar synthesis method as that of Intermediate 1-2.
The synthesis steps refer to Step 1 of Example 5. Intermediate 1-17 (2.23 g, yield of 92%) is prepared from 5-methyl-4-(1-methyl-1H-pyrazol-5-yl)thiazole-2-formaldehyde (Intermediate 1-16) (2.07 g, 10.0 mmol) with the similar synthesis method as that of Intermediate 1-8.
The synthesis steps refer to Step 2 of Example 4. Intermediate 10 (2.2 g, yield of 85%) is prepared from 5-methyl-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)thiazole-2-formaldehyde (Intermediate 1-17) (2.4 g, 10.0 mmol) with the similar synthesis method as that of Intermediate 5.
The synthesis steps refer to Step 1 of Example 1. Intermediate 1-19 (1.88 g, yield of 86%) is prepared from 5-methyl-4-bromopyrrole-2-methyl formate (Compound 1-18) (2.2 g, 10.0 mmol) with the similar synthesis method as of Compound 1-2.
The synthesis steps refer to Step 1 of Example 3. Intermediate 1-20 (2.37 g, yield of 79%) is prepared from 5-methyl-4-(1-methyl-1H-pyrazol-5-yl)pyrrole-2-methyl formate (Intermediate 1-19) (2.19 g, 10.0 mmol) with the similar synthesis method as that of Intermediate 1-5.
The synthesis steps refer to Step 2 of Example 1. Intermediate 11 (2.57 g, yield of 86%) is prepared from 5-methyl-4-(4-bromo-1-methyl-1H-pyrazol-5-yl)pyrrole-2-methyl formate (Intermediate 1-20) (2.98 g, 10.0 mmol) with the similar synthesis method as that of Intermediate 1.
The synthesis steps refer to Step 1 of Example 1. Intermediate 1-22 (1.9 g, yield of 77%) is prepared from 1-methyl-5-ethyl-4-bromopyrrole-2-methyl formate (Compound 1-21) (2.5 g, 10.1 mmol) with the similar synthesis methods as that of Intermediate 1-2.
The synthesis steps refer to Step 1 of Example 5. Intermediate 1-23 (2.45 g, yield of 87%) is prepared from 1-methyl-5-ethyl-4-(1-methyl-1H-pyrazol-5-yl)pyrrole-2-methyl formate (Intermediate 1-22) (2.5 g, 10.2 mmol) with the similar synthesis method as that of Intermediate 1-8.
The synthesis steps refer to Step 2 of Example 1. Intermediate 12 (2.44 g, yield of 93%) is prepared from 1-methyl-5-ethyl-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)pyrrole-2-methyl formate (Intermediate 1-23) (2.8 g, 10.0 mmol) with the similar synthesis method as that of Intermediate 1.
The synthesis steps refer to Step 1 of Example 1. Intermediate 1-25 (1.92 g, yield of 87%) is prepared from 4-bromopyrrole-2-methyl formate (Compound 1-24) (2.0 g, 9.9 mmol) with the similar synthesis method as that of Intermediate 1-2.
The synthesis steps refer to Step 1 in Example 3. Intermediate 1-26 (2.57 g, yield of 86%) is prepared from 4-(1,4-dimethyl-1H-pyrazol-5-yl)pyrrole-2-methyl formate (Intermediate 1-25) (2.19 g, 10.0 mmol) with the similar synthesis method as that of Intermediate 1-5.
The synthesis steps refer to Step 2 of Example 1. Intermediate 13 (2.6 g, yield of 91%) is prepared from 5-bromo-4-(1,4-dimethyl-1H-pyrazol-5-yl)pyrrole-2-methyl formate (Intermediate 1-26) (2.99 g, 10.0 mmol) with the similar synthesis method as that of Intermediate 1.
The synthesis steps refer to Step of in Example 1. Intermediate 1-28 (2.54 g, yield of 92%) is prepared from 1-methyl-5-ethyl-4-bromopyrrole-2-methyl formate (Compound 1-27) (2.3 g, 9.9 mmol) with the similar synthesis method as that of Intermediate 1-2.
The synthesis steps refer to Step 2 of Example 1. Intermediate 14 (2.25 g, yield of 86%) is prepared from 1-methyl-5-ethyl-4-(4-ethyl-1-methyl-1H-pyrazol-5-yl)pyrrole-2-methyl formate (Intermediate 1-28) (2.8 g, 10.2 mmol) with the similar synthesis method as that of Intermediate 1.
The synthesis steps refer to Step 1 of Example 1. Intermediate 1-30 (1.76 g, yield of 85%) is prepared from 4-bromo-oxazole-2-methyl formate (Compound 1-29) (2.03 g, 9.9 mmol) with the similar synthesis method as that of Intermediate 1-2.
The synthesis steps refer to Step 2 in Example 1. Intermediate 15 (1.85 g, yield of 95%) is prepared from 4-(1-methyl-1H-pyrazol-5-yl)oxazole-2-methyl formate (Intermediate 1-30) (2.09 g, 10.2 mmol) with the similar synthesis method as that of Intermediate 1.
The synthesis steps refer to Step 1 of Example 1. Intermediate 1-31 (1.8 g, yield of 87%) is prepared from 4-bromofuran-2-methyl formate (Compound 1-1) (2.08 g, 10.3 mmol) with the similar synthesis methods as that of Intermediate 1-2.
The synthesis steps refer to Step 1 of Example 3. Intermediate 1-32 (2.43 g, yield of 85%) is prepared from 4-(1-methyl-1H-1,2,4-triazol-5-yl)furan-2-methyl formate (Intermediate 1-31) (2.1 g, 10.1 mmol) with the similar synthesis method as that of Intermediate 1-5.
The synthesis steps refer to Step 2 of Example 1. Intermediate 16 (2.54 g, yield of 93%) is prepared from 5-bromo-4-(1-methyl-1H-1,2,4-triazol-5-yl)furan-2-methyl formate (Intermediate 1-32) (2.90 g, 10.2 mmol) with the similar synthesis method as that of Intermediate 1.
The synthesis steps refer to Step 1 of Example 1. Intermediate 1-34 (2.12 g, yield of 85%) is prepared from 1-methyl-3-ethyl-5-bromo-4-bromofuran-2-methyl formate (Compound 1-33) (2.5 g, 10.2 mmol) with the similar synthesis method as that of Intermediate 1-2.
The synthesis steps refer to Step 1 of Example 3. Intermediate 1-35 (3.03 g, yield of 93%) is prepared from 1-methyl-3-ethyl-4-(1-methyl-1H-1,2,4-triazol-5-yl)pyrrole-2-methyl formate (Intermediate 1-34) (2.5 g, 10.1 mmol) with the similar synthesis method as that of Intermediate 1-5.
The synthesis steps refer to Step 2 of Example 1. Intermediate 17 (2.99 g, yield of 96%) is prepared from 1-methyl-3-ethyl-5-bromo-4-(1-methyl-1H-1,2,4-triazol-5-yl)pyrrole-2-methyl formate (Intermediate 1-35) (3.3 g, 10.2 mmol) with the similar synthesis method as that of Intermediate 1.
The synthesis steps refer to Step 1 of Example 1. Intermediate 1-37 (1.7 g, yield of 82%) is prepared from 3-methyl-4-bromofuran-2-formaldehyde (Compound 1-36) (2.2 g, 10.2 mmol) with the similar synthesis methods as that of Intermediate 1-2.
The synthesis steps refer to Step 1 of Example 5. Intermediate 1-38 (2.13 g, yield of 88%) is prepared from 3-methyl-4-(1-methyl-1H-1,2,4-triazol-5-yl)thiophene-2-formaldehyde (Intermediate 1-37) (2.1 g, 10.2 mmol) with the similar synthesis method as that of Compound 1-8.
The synthesis steps refer to Step 2 of Example 4. Intermediate 18 (2.37 g, yield of 92%) is prepared from 3-methyl-5-chloro-4-(1-methyl-1H-1,2,4-triazol-5-yl)thiophene-2-formaldehyde (Intermediate 1-38) (2.4 g, 10.0 mmol) with the similar synthesis method as that of Intermediate 5.
The synthesis steps refer to Step 1 of Example 1. Intermediate 1-40 (1.82 g, yield of 76%) is prepared from 4-chloro-5-bromofuran-2-methyl formate (Compound 1-39) (2.43 g, 10.4 mmol) with the similar synthesis method as that of Intermediate 1-2.
The synthesis steps refer to Step 2 of Example 1. Intermediate 19 (2.01 g, yield of 88%) is prepared from 4-chloro-5-(1-methyl-1H-1,2,4-triazol-5-yl)furan-2-methyl formate (Intermediate 1-40) (2.49 g, 10.2 mmol) with the similar synthesis method as that of Intermediate 1.
The synthesis steps refer to Step 1 of Example 1. Intermediate 1-43 (1.89 g, yield of 83%) is prepared from 4-chloro-5-bromothiophene-2-formaldehyde (Compound 1-42) (2.3 g, 10.4 mmol) with the similar synthesis method as that of Intermediate 1-2.
The synthesis steps refer to Step 2 of Example 4. Intermediate 20 (2.27 g, yield of 93%) is prepared from 4-chloro-5-(1-methyl-1H-1,2,4-triazol-5-yl)thiophene-2-formaldehyde (Intermediate 1-43) (2.3 g, 10.2 mmol) with the similar synthesis method as that of Intermediate 5.
The synthesis steps refer to Step 1 of Example 1. Intermediate 1-45 (1.69 g, yield of 74%) is prepared from 4-methyl-5-bromothiophene-2-formaldehyde (Compound 1-44) (2.1 g, 10.4 mmol) with the similar synthesis method as that of Intermediate 1-2.
The synthesis steps refer to Step 2 of Example 1. Intermediate 21 (2.01 g, yield of 88%) is prepared from 4-methyl-5-(1-methyl-1H-1,2,4-triazol-5-yl) furan-2-methyl formate (Intermediate 1-45) (2.49 g, 10.2 mmol) with the similar synthesis method as that of Intermediate 1.
The synthesis steps refer to Step 1 of Example 1. Intermediate 1-46 (1.9 g, yield of 79%) is prepared from 4-chloro-5-bromofuran-2-methyl formate (Compound 1-39) (2.5 g, 10.4 mmol) with the similar synthesis method as that of Intermediate 1-2.
The synthesis steps refer to Step 1 of Example 3. Intermediate 1-47 (2.87 g, yield of 90%) is prepared from 4-chloro-5-(1-methyl-1H-pyrazol-5-yl)furan-2-methyl formate (Intermediate 1-46) (2.48 g, 10.1 mmol) with the similar synthesis method as that of Intermediate 1-5.
The synthesis steps refer to Step 2 of Example 1. Intermediate 22 (2.68 g, yield of 88%) is prepared from 4-chloro-5-(4-bromo-1-methyl-1H-pyrazol-5-yl) furan-2-methyl formate (Intermediate 1-47) (3.22 g, 10.2 mmol) with the similar synthesis method as that of Intermediate 1.
Dissolving Compound 2-1b (2.96 g, 11.62 mmol) in anhydrous tetrahydrofuran (20 ml), under the protection of N2, slowly dropwise adding 2N borane dimethyl sulfide solution within tetrahydrofuran (3.29 ml, 34.88 mmol) thereto under an ice bath, stirring for about 3 h at room temperature. Monitoring the reaction with TLC thin-layer chromatography for whether it is completed, after the reaction, under the ice bath, slowly dropwise adding methanol into the reaction liquid until no air bubbles. Then adding about 3 ml of 1N HCl solution into the reaction liquid, stirring for 10 min at room temperature, recycling the solvent under reduced pressure, and washing with saturated NaHCO3 solution, followed by extracting the reaction liquid with ethyl acetate 3 times, washing the merged organic phase with saturated sodium chloride once, and drying it with anhydrous sodium sulfate. Recycling the solvent under reduced pressure, 2.10 g of light yellow oily liquid (Intermediate 2-2b) is obtained and the yield is 75%.
Dissolving Intermediate 2-2b (2.10 g, 6.36 mmol) into the mixed solution of anhydrous ethanol (10 ml) and glacial acetic acid (10 ml), adding zinc powder (1.65 g, 25.44 mmol) thereto under an ice bath, and stirring overnight at room temperature under the protection of N2. After the reaction is completed, suction filtrating the reaction mixture, neutralizing the glacial acetic acid in the filtrate with saturated Na2CO3 to a pH of greater than 7, extracting the reaction liquid with ethyl acetate 3 times, washing with saturated sodium chloride once, and drying it with anhydrous sodium sulfate. Recycling the solvent under reduced pressure, 1.55 g of oily liquid (Intermediate 24) is obtained and the yield is 81%.
The synthesis steps refer to Step 1 of Example 1. Intermediate 3-2 (1.98 g, yield of 85%) is prepared from 6-methyl-4-bromopyridinyl-2-methyl formate (Compound 3-1) (2.2 g, 9.9 mmol) with the similar synthesis method as that of Intermediate 1-2.
The synthesis steps refer to Step 1 of Example 5. Intermediate 3-3 (1.89 g, yield of 83%) is prepared from 6-methyl-4-(1-methyl-1H-pyrazol-5-yl)pyridinyl-2-methyl formate (Intermediate 3-2) (1.98 g, 8.6 mmol) with the similar synthesis method as that of Intermediate 1-8.
The synthesis steps refer to Step 2 of Example 1. Intermediate 41 (1.48 g, yield of 82%) is prepared from 6-methyl-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)pyridinyl-2-methyl formate (Intermediate 3-3) (1.89 g, 7.1 mmol) with the similar synthesis method as that of Intermediate 1.
The synthesis steps refer to Step 1 of Example 1. Intermediate 3-5 (2.58 g, yield of 86%) is prepared from 4-fluoro-5-bromopyridinyl-2-methyl formate (Compound 3-4) (2.8 g, 11.9 mmol) with the similar synthesis method as that of Intermediate 1-2.
The synthesis steps refer to Step 2 of Example 1. Intermediate 42 (2.08 g, yield of 86%) is prepared from 4-fluoro-5-(1,4-dimethyl-1H-pyrazol-5-yl)pyridinyl-2-methyl formate (Intermediate 3-5) (2.58 g, 10.4 mmol) with the similar synthesis method as that of Intermediate 1.
The synthesis steps refer to Step 1 of Example 1. Intermediate 3-7 (2.28 g, yield of 86%) is prepared from 2-methyl-6-bromopyrimidine-4-methyl formate (Compound 3-6) (2.35 g, 10.2 mmol) with the similar synthesis method as that of Intermediate 1-2.
The synthesis steps refer to Step 2 of Example 1. Intermediate 43 (1.88 g, yield of 87%) is prepared from 2-methyl-6-(1-methyl-4-ethyl-1H-pyrazol-5-yl)pyrimidine-4-methyl formate (Intermediate 3-7) (2.28 g, 8.76 mmol) with the similar synthesis method as that of Intermediate 1.
The synthesis steps refer to Step 1 of Example 1. Intermediate 3-9 (2.28 g, yield of 86%) is prepared from 2-methyl-6-bromopyrimidine-4-methyl formate (Compound 3-8) (2.35 g, 10.2 mmol) with the similar synthesis method as that of Intermediate 1-2.
The synthesis steps refer to Step 1 of Example 3. Intermediate 3-10 (2.57 g, yield of 86%) is prepared from 5-(1-methyl-1H-pyrazol-5-yl)pyrazine-2-methyl formate (Compound 3-9) (2.19 g, 10.0 mmol) with the similar synthesis method as that of Intermediate 1-5.
The synthesis steps refer to Step 2 of Example 1. Intermediate 44 (1.88 g, yield of 87%) is prepared from 5-(4-bromo-1-methyl-1H-pyrazol-5-yl)pyrazine-2-methyl formate (Intermediate 3-10) (2.28 g, 8.76 mmol) with the similar synthesis method as that of Intermediate 1.
The synthesis steps refer to Step 1 of Example 1. Intermediate 3-12 (2.18 g, yield of 87%) is prepared from 5-ethyl-6-bromopyridinyl-2-methyl formate (Compound 3-11) (2.49 g, 10.2 mmol) with the similar synthesis method as that of Intermediate 1-2.
The synthesis steps refer to Step 1 of Example 3. Intermediate 3-13 (2.46 g, yield of 85%) is prepared from 5-ethyl-6-(1-methyl-1H-pyrazol-5-yl)pyridinyl-2-methyl formate (Intermediate 3-12) (2.18 g, 8.9 mmol) with the similar synthesis method as that of Intermediate 1-5.
The synthesis steps refer to Step 2 of Example 1. Intermediate 45 (1.98 g, yield of 84%) is prepared from 5-ethyl-6-(4-bromo-1-methyl-1H-pyrazol-5-yl)pyridinyl-2-methyl formate (Intermediate 3-13) (2.46 g, 7.6 mmol) with the similar synthesis method as that of Intermediate 1.
The synthesis steps refer to Step 1 of Example 1. Intermediate 3-15 (2.17 g, yield of 87%) is prepared from 4,6-dimethyl-5-bromopyrimidine-2-methyl formate (Compound 3-14) (2.49 g, 10.2 mmol) with the similar synthesis method as that of Intermediate 1-2.
The synthesis steps refer to Step 1 of Example 3. Intermediate 3-16 (2.46 g, yield of 85%) is prepared from 4,6-dimethyl-5-(1-methyl-1H-pyrazol-5-yl)pyrimidine-2-methyl formate (Intermediate 3-15) (2.17 g, 8.9 mmol) with the similar synthesis method as that of Intermediate 1-5.
The synthesis steps refer to Step 2 of Example 1. Intermediate 46 (1.98 g, yield of 84%) is prepared from 4,6-dimethyl-5-(4-bromo-1-methyl-1H-pyrazol-5-yl)pyrimidine-2-methyl formate (Intermediate 3-16) (2.46 g, 7.6 mmol) with the similar synthesis method as that of Intermediate 1.
The synthesis steps refer to Step 1 of Example 1. Intermediate 3-18 (2.67 g, yield of 89%) is prepared from 5-chloro-4-bromopyrimidine-2-methyl formate (Compound 3-17) (2.57 g, 10.2 mmol) with the similar synthesis method as that of Intermediate 1-2.
The synthesis steps refer to Step 2 of Example 1. Intermediate 47 (2.33 g, yield of 92%) is prepared from 5-chloro-4-(4-isopropyl-1-methyl-1H-pyrazol-5-yl)pyrimidine-2-methyl formate (Intermediate 3-18) (2.67 g, 9.1 mmol) with the similar synthesis method as that of Intermediate 1.
The synthesis steps refer to Step 1 of Example 1. Intermediate 3-20 (2.11 g, yield of 89%) is prepared from 4-methyl-5-bromopyrimidine-2-methyl formate (Compound 3-19) (2.35 g, 10.2 mmol) with the similar synthesis method as that of Intermediate 1-2.
The synthesis steps refer to Step 1 of Example 5. Intermediate 3-21 (2.17 g, yield of 90%) is prepared from 4-methyl-5-(1-methyl-1H-pyrazol-5-yl)pyrimidine-2-methyl formate (Intermediate 3-20) (2.11 g, 9.0 mmol) with the similar synthesis method as that of Intermediate 1-8.
The synthesis steps refer to Step 2 of Example 1. Intermediate 48 (1.76 g, yield of 86%) is prepared from 4-methyl-5-(4-chloro-1-methyl-1H-pyrazol-5-yl)pyrimidine-2-methyl formate (Intermediate 3-21) (2.17 g, 8.1 mmol) with the similar synthesis method as that of Intermediate 1.
The synthesis steps refer to Step 1 of Example 1. Intermediate 3-23 (2.54 g, yield of 90%) is prepared from 2-chloro-3-bromophenyl methyl formate (Compound 3-22) (2.53 g, 10.2 mmol) with the similar synthesis method as that of Intermediate 1-2.
The synthesis steps refer to Step 2 of Example 1. Intermediate 49 (2.23 g, yield of 92%) is prepared from 2-chloro-3-(4-ethyl-1-methyl-1H-pyrazol-5-yl)phenyl methyl formate (Intermediate 3-23) (2.54 g, 9.1 mmol) with the similar synthesis method as that of Intermediate 1.
The synthesis steps refer to Step 1 of Example 1. Intermediate 3-25 (2.38 g, yield of 79%) is prepared from 3-n-propyl-4-bromophenyl methyl formate (Compound 3-24) (3.0 g, 11.7 mmol) with the similar synthesis method as that of Intermediate 1-2.
The synthesis steps refer to Step 1 of Example 3. Intermediate 3-26 (2.67 g, yield of 88%) is prepared from 3-n-propyl-4-(1-methyl-1H-pyrazol-5-yl)phenyl methyl formate (Intermediate 3-25) (2.38 g, 9.22 mmol) with the similar synthesis method as that of Intermediate 1-5.
The synthesis steps refer to Step 2 of Example 1. Intermediate 50 (2.34 g, yield of 92%) is prepared from 3-n-propyl-4-(4-bromo-1-methyl-1H-pyrazol-5-yl)phenyl methyl formate (Intermediate 3-26) (2.67 g, 7.9 mmol) with the similar synthesis method as that of Intermediate 1.
The synthesis steps refer to Step 1 of Example 1. Intermediate 3-28 (2.2 g, yield of 88%) is prepared from 4-chloro-5-bromopyrimidine-2-methyl formate (Compound 3-27) (2.5 g, 9.9 mmol) with the similar synthesis method as that of Compound 1-2.
The synthesis steps refer to Step 1 of Example 5. Intermediate 3-29 (2.14 g, yield of 85%) is prepared from 4-chloro-5-(1H-pyrazol-5-yl)pyrimidine-2-methyl formate (Intermediate 3-28) (2.2 g, 8.7 mmol) with the similar synthesis method as that of Intermediate 1-8.
The synthesis steps refer to Step 2 of Example 1. Intermediate 51 (1.89 g, yield of 93%) is prepared from 4-chloro-5-(4-ch*/6loro-1H-pyrazol-5-yl)pyrimidine-2-methyl formate (Intermediate 3-29) (2.14 g, 7.4 mmol) with the similar synthesis method as that of Intermediate 1.
The synthesis steps refer to Step 1 of Example 1. Intermediate 3-31 (1.47 g, yield of 81%) is prepared from 4-methyl-5-bromopyridinyl-2-methyl formate (Compound 3-30) (1.8 g, 7.8 mmol) with the similar synthesis method as that of Intermediate 1-2.
The synthesis steps refer to Step 1 of Example 5. Intermediate 3-32 (1.3 g, yield of 77%) is prepared from 4-methyl-5-(1H-pyrazol-5-yl)pyridinyl-2-methyl formate (Intermediate 3-31) (1.47 g, 6.3 mmol) with the similar synthesis method as that of Intermediate 1-8.
The synthesis steps refer to Step 2 of Example 1. Intermediate 52 (0.9 g, yield of 73%) is prepared from 4-methyl-5-(4-chloro-1H-pyrazol-5-yl)pyridinyl-2-methyl formate (Intermediate 3-32) (1.3 g, 4.9 mmol) with the similar synthesis method as that of Intermediate 1.
The synthesis steps refer to Step 1 of Example 1. Intermediate 3-34 (2.8 g, yield of 90%) is prepared from 2,5-dimethyl-4-bromophenyl methyl formate (Compound 3-33) (3.1 g, 12.7 mmol) with the similar synthesis method as that of Intermediate 1-2.
The synthesis steps refer to Step 1 of Example 3. Intermediate 3-35 (3.18 g, yield of 86%) is prepared from 2,5-dimethyl-4-(1-methyl-1H-pyrazol-5-yl)phenyl methyl formate (Intermediate 3-34) (2.8 g, 11.5 mmol) with the similar synthesis method as that of Intermediate 1-5.
The synthesis steps refer to Step 2 of Example 1. Intermediate 53 (2.7 g, yield of 87%) is prepared from 2,5-dimethyl-4-(4-bromo-1-methyl-1H-pyrazol-5-yl)phenyl methyl formate (Intermediate 3-35) (3.18 g, 9.8 mmol) with the similar synthesis method as that of Intermediate 1.
The synthesis steps refer to Step 1 of Example 1. Intermediate 3-37 (2.1 g, yield of 84%) is prepared from 6-methyl-5-bromopyridinyl-2-methyl formate (Compound 3-36) (2.5 g, 10.8 mmol) with the similar synthesis method as that of Intermediate 1-2.
The synthesis steps refer to Step 1 of Example 3. Intermediate 3-38 (2.27 g, yield of 81%) is prepared from 6-methyl-5-(1-methyl-1H-pyrazol-5-yl)pyridinyl-2-methyl formate (Intermediate 3-37) (2.1 g, 9.1 mmol) with the similar synthesis method as that of Intermediate 1-5.
The synthesis steps refer to Step 2 of Example 1. Intermediate 54 (1.9 g, yield of 88%) is prepared from 6-methyl-5-(4-bromo-1-methyl-1H-pyrazol-5-yl)pyridinyl-2-methyl formate (Intermediate 3-38) (2.27 g, 7.3 mmol) with the similar synthesis method as that of Intermediate 1.
The synthesis steps refer to Step 1 of Example 1. Intermediate 3-40 (2.0 g, yield of 89%) is prepared from 4-methyl-5-bromopyrimidine-2-methyl formate (Compound 3-39) (2.1 g, 9.1 mmol) with the similar synthesis method as that of Intermediate 1-2.
The synthesis steps refer to Step 2 of Example 1. Intermediate 55 (1.4 g, yield of 74%) is prepared from 4-methyl-5-(1,4-dimethyl-1H-pyrazol-5-yl)pyrimidine-2-methyl formate (Intermediate 3-40) (2.0 g, 8.1 mmol) with the similar synthesis method as that of Intermediate 1.
Dissolving 2-nitroethyl tert-butyl carbamate (Compound 6-5, 380 mg, 2 mmol), ((S)-(−)-α,α-diphenyl-2-pyrrylmethyl)trimethylsilyl ether (33 mg, 0.1 mmol), and benzoic acid (25 mg, 0.2 mmol) into anhydrous dichloromethane (2 ml), slowly adding Intermediate 4-1a (201 mg, 1 mmol) thereto in an ice bath under the protection of N2, stirring for about 24 h at room temperature, diluting the reaction system with dichloromethane to 10 ml, slowly dropwise adding 200 μl of trifluoroacetic acid into the reaction liquid in an ice bath, and reacting for 5 h at room temperature. Then adding about 10 ml of 1N NaHCO3 solution into the reaction liquid, stirring for 10 min at room temperature, followed by extracting the reaction liquid with ethyl acetate 3 times, washing the merged organic phase with saturated sodium chloride once, drying it with anhydrous sodium sulfate, and purifying by column chromatograph, 270 mg of light yellow oily liquid (Intermediate 4-2a) is obtained and the yield is 72%.
Dissolving Intermediate 4-2a (186 mg, 0.5 mmol) into methanol (10 ml), adding 30 mg of 10% Pd/C thereto, and hydrogenating overnight at room temperature (monitoring the reaction with TLC thin-layer chromatography for whether is completed). After the reaction is completed, filtering to remove black insoluble substance from the reaction mixture, and spin drying under reduced pressure, 120 mg of oily liquid (Intermediate 56) is obtained and the yield is 70%.
Dissolving Compound 5-1b of 3,4-difluorobenzaldehyde (5.5 ml, 50 mmol), nitromethane (22.5 ml, 420 mmol), and ammonium acetate (9.85 g, 128 mmol) in glacial acetic acid (70 ml), heating to 90° C. and reacting for 3 h, monitoring the reaction with TLC thin-layer chromatography for whether it is completed. Adding water (20 ml) into the reaction liquid after the reaction is completed, neutralizing glacial acetic acid in the reaction liquid with Na2CO3 to a pH of about 7, extracting the reaction liquid with ethyl acetate 3 times, washing the merged organic phase with saturated sodium chloride once, and drying it with anhydrous sodium sulfate. Recycling the solvent under reduced pressure. Carrying out column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=10:1), 7.0 g of yellow solid (Intermediate 5-2b) is obtained. The yield is 85%.
Synthesis steps of Intermediate 5-2a (21 g, yield of 80%) refer to the synthesis method of Compound 5-2b in Step 1 of Example 39, in which Intermediate 5-2a is prepared from 4-trifluoromethylbenzaldehyde (Compound 5-1a) (20 g, 114.9 mmol).
Dissolving Intermediate 5-2b (501 mg, 2.71 mmol) and trifluoroacetic acid (0.02 mL, 0.271 mmmol) into dichloromethane (10 ml), slowly dropwise adding dichloromethane solution (10 ml) dissolved with N-methoxymethyl-N-(trimethylsilane methyl)benzylamine (1.0 ml, 5.42 mmol) thereto at 0° C. under the protection of N2, and stirring overnight at room temperature. After the reaction is completed, adding 10 ml of water to the reaction liquid, extracting the reaction liquid with ethyl acetate 3 times, washing the merged organic phase with saturated sodium chloride once, and drying it with anhydrous sodium sulfate. Recycling the solvent under reduced pressure. Purifying by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=9:1), 603 mg of yellow-green semi-solid (Intermediate 5-3b) is obtained. The yield is 81%.
Synthesis steps of Intermediate 5-3a refer to that of Compound 5-3b in Step 2 of Example 39, in which Intermediate 5-3a (14.5 g, yield of 44%) is prepared from Intermediate 5-2a (21 g, 90.5 mmol).
Dissolving Intermediate 5-3b (302 mg, 0.95 mmol) and anhydrous stannous chloride (1.07 mg, 4.75 mmmol) into ethyl acetate (10 ml), raising temperature to 50° C. and reacting for 2 h. After the reaction is completed, adding saturated NaHCO3 solution (10 ml) to the reaction liquid, extracting the reaction liquid with ethyl acetate 3 times, washing the merged organic phase with saturated sodium chloride once, and drying it with anhydrous sodium sulfate. Recycling the solvent under reduced pressure. Purifying by column chromatography on silica gel (eluent: petroleum ether:ethyl acetate=9:1, and then increase the polarity to ethyl acetate:methanol=20:1), 123 mg of oily liquid (Intermediate 63) is obtained and the yield is 61%.
Synthesis steps of Intermediate 62 refer to that of Intermediate 63 in Step 3 of Example 39, in which Intermediate 62 (8.6 g, yield of 68%) is prepared from Intermediate 5-3a (14.5 g, 39.7 mmol).
Dissolving 2-nitroethyl tert-butyl carbamate (Compound 6-5, 2.85 g, 15 mmol), ((S)-(−)-α,α-diphenyl-2-pyrrylmethyl)trimethylsilyl ether (0.36 g, 1.1 mmol), and benzoic acid (0.25 g, 2 mmol) in anhydrous dichloromethane (15 ml), slowly adding Compound 4-1a (2.01 g, 10 mmol) in an ice bath under the protection of N2, stirring for about 18 h at room temperature, and diluting the reaction system with dichloromethane to 100 ml. Adding allyltrimethylsilane (5 ml, 30 mmol) into the reaction liquid in an ice bath, decreasing the temperature of the reaction system to −78° C., slowly dropwise adding 2.5 ml of aether boron trifluoride, continue to react for 10 h. Adding about 100 ml of 1N NaHCO3 solution into the reaction liquid, stirring for 10 min at room temperature, and then extracting the reaction liquid with ethyl acetate 3 times, washing the merged organic phase with saturated sodium chloride once, and drying it with anhydrous sodium sulfate. Passing through silica gel column, 2.1 g of white solid (Intermediate 64) is obtained and the yield is 50.9%; 1H NMR (500 MHz, CDCl3) δ7.39 (d, J=8.3 Hz, 1H), 7.29 (d, J=1.7 Hz, 1H), 7.04 (dd, J=8.3, 1.7 Hz, 1H), 5.81-5.67 (m, 1H), 5.16 (d, J=17.1 Hz, 1H), 5.10 (d, J=9.9 Hz, 1H), 4.77-4.35 (m, 3H), 3.46 (dd, J=17.1, 11.2 Hz, 1H), 3.38-3.20 (m, 1H), 2.63-2.51 (m, 1H), 2.46-2.31 (m, 1H), 1.96-1.82 (m, 2H), 1.48 (s, 9H).
Dissolving Intermediate 64 (2.10 g, 5 mmol) into the mixed solution of anhydrous ethanol (40 ml) and water (10 ml), adding iron powder (2.3 g, 40 mmol), and ammonium chloride (0.8, 15 mmol) thereto, after the protection of N2, reacting for 2 h under heating reflux with mechanical stirring, performing suction filtration and spin dry, adding 60 ml of saturated Na2CO3 solution, washing with ethyl acetate 3 times and then with saturated sodium chloride once, and drying it with anhydrous sodium sulfate. Recycling the solvent under reduced pressure, 1.6 g of light yellow solid (Intermediate 65) is obtained and the yield is 83.1%.
Dissolving Intermediate 65 (0.82 g, 3.14 mmol), 1-hydroxybenzotriazole (HOBT) (0.76 g, 5.65 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDC.HCl (1.1 g, 5.65 mmol) in anhydrous dichloromethane (50 ml), after stirring for 10 min in an ice bath, adding diisopropylethylamine (1.4 ml, 7.85 mmol), continue stirring for 30 min in an ice bath, slowly adding dichloromethane solution (10 ml) dissolved with Intermediate 3 (1.2 g, 3.14 mmol), and stirring overnight at room temperature. Pouring the reaction liquid to 100 ml of saturated Na2CO3 solution, extracting the reaction liquid with dichloromethane 3 times, washing the merged organic phase with saturated sodium chloride once, drying it with anhydrous sodium sulfate, recycling the solvent under reduced pressure, carrying out column chromatography on silica gel, 1.43 g of white powder (Intermediate 66) is obtained and the yield is 73.2%; 1H NMR (500 MHz, CDCl3) δ7.48 (s, 1H), 7.38 (d, J=8.2 Hz, 1H), 7.34 (d, J=1.8 Hz, 1H), 7.12 (d, J=8.2 Hz, 2H), 6.04 (s, 1H), 5.84-5.71 (m, 1H), 5.14 (d, J=17.0 Hz, 1H), 5.08 (d, J=10.1 Hz, 1H), 4.57-4.27 (m, 2H), 4.26-4.16 (m, 1H), 3.76 (s, 3H), 3.12-2.94 (m, 1H), 2.89 (t, J=12.0 Hz, 1H), 2.58-2.47 (m, 1H), 2.45-2.30 (m, 1H), 1.94-1.79 (m, 2H), 1.48 (s, 9H).
Dissolving Intermediate 66 (280 mg, 0.45 mmol) in the mixed solution of tetrahydrofuran (12 ml) and water (4 ml), adding N-methylmorpholine nitrogen oxide (105 mg, 9 mmol) and osmium tetroxide (6 mg, 0.02 mmol) thereto, stirring overnight at room temperature, pouring the reaction liquid into 30 ml of saturated sodium thiosulfate solution, extracting the reaction liquid with ethyl acetate 3 times, washing with saturated sodium chloride once, drying it with anhydrous sodium sulfate, 288.1 mg of white solid (Intermediate 67) is obtained by spin drying and the yield is 98.2%; 1H NMR (500 MHz, CDCl3) δ7.49 (s, 1H), 7.40 (d, J=8.2 Hz, 1H), 7.35 (s, 1H), 7.16-7.10 (m, 2H), 6.00 (d, J=6.9 Hz, 1H), 4.68-4.61 (m, 1H), 4.41-4.33 (m, 1H), 4.30-4.20 (m, 1H), 3.77 (s, 3H), 3.67-3.47 (m, 3H), 3.04-2.94 (m, 1H), 2.86-2.76 (m, 1H), 2.11-2.00 (m, 2H), 1.83 (d, J=13.6 Hz, 2H), 1.50 (s, 9H).
Dissolving Intermediate 67 (288 mg, 0.43 mmol) in the mixed solution of tetrahydrofuran (6 ml) and water (2 ml), adding sodium periodate (171 mg, 0.8 mmol) thereto, stirring for 2 h at room temperature, adding 10 ml of saturated sodium chloride solution into the reaction liquid for diluting, extracting the reaction liquid with ethyl acetate 3 times, merging the organic layer, and drying it with anhydrous sodium sulfate, 257 mg of white solid (Intermediate 68) is obtained after spin drying, and the yield is 93.5%; 1H NMR (500 MHz, CDCl3) δ9.79 (s, 1H), 7.49 (s, 1H), 7.39 (d, J=8.0 Hz, 1H), 7.34 (d, J=1.8 Hz, 1H), 7.15-7.10 (m, 2H), 6.11 (s, 1H), 5.13-4.86 (m, 1H), 4.54-4.28 (m, 1H), 4.27-4.15 (m, 1H), 3.76 (s, 3H), 3.11-2.86 (m, 3H), 2.75-2.69 (m, 1H), 2.03-1.97 (m, 1H), 1.88-1.85 (m, 1H), 1.48 (s, 9H).
Dissolving Intermediate 68 (200 mg, 0.32 mmol) in the mixed solution of tetrahydrofuran (5 ml) and water (0.5 ml), slowly adding sodium borohydride (24 mg, 0.64 mmol) under an ice bath, reacting for 2 h at room temperature, slowly dropwise adding 6 ml of saturated ammonium chloride solution to the reaction liquid, extracting the reaction liquid with ethyl acetate 3 times, merging the organic layer, washing with saturated sodium chloride twice, and drying it with anhydrous sodium sulfate, 183 mg of white solid (Intermediate 69) is obtained after spin drying, and the yield is 92.6%; 1H NMR (500 MHz, CDCl3) δ7.49 (s, 1H), 7.40 (d, J=8.3 Hz, 1H), 7.36 (d, J=1.6 Hz, 1H), 7.16-7.11 (m, 2H), 6.01 (d, J=8.0 Hz, 1H), 4.66-4.58 (m, 1H), 4.40-4.32 (m, 1H), 4.28-4.17 (m, 1H), 3.76 (s, 3H), 3.69 (d, J=11.3 Hz, 1H), 3.41 (t, J=11.3 Hz, 1H), 2.99 (t, J=11.0 Hz, 1H), 2.81 (t, J=12.1 Hz, 1H), 2.11-2.00 (m, 3H), 1.84 (d, J=12.1 Hz, 1H), 1.71 (d, J=12.1 Hz, 1H), 1.50 (s, 9H).
Dissolving Intermediate 69 (100 mg, 0.16 mmol) in anhydrous dichloromethane (5 ml), adding diisopropylethylamine (0.083 ml, 0.48 mmol) and methylsulfonyl chloride (0.031 ml, 0.40 mmol) under an ice bath, and reacting for 2 h at room temperature. Adding 15 ml of saturated NaHCO3 solution to the reaction liquid, extracting thereof with dichloromethane twice, merging the organic layer, washing with saturated sodium chloride twice, and drying it with anhydrous sodium sulfate, 92 mg of white solid (Intermediate 70) is obtained after spin drying and carrying out column chromatography, and the yield is 81.2%; 1H NMR (500 MHz, CDCl3) δ7.49 (s, 1H), 7.39 (d, J=8.3 Hz, 1H), 7.36 (d, J=1.9 Hz, 1H), 7.15-7.12 (m, 2H), 6.06 (s, 1H), 4.67-4.33 (m, 2H), 4.33-4.22 (m, 2H), 4.22-4.12 (m, 1H), 3.77 (s, 3H), 3.14-2.96 (m, 4H), 2.91 (t, J=12.2 Hz, 1H), 2.41-2.30 (m, 1H), 2.02-1.91 (m, 2H), 1.85 (d, J=12.6 Hz, 1H), 1.50 (s, 9H).
Dissolving 4-(1-methyl-1H-pyrazol-5-yl)furan-2-formic acid (Intermediate 1, 66.2 mg, 0.345 mmol), 1-hydroxybenzotriazole (HOBT) (78.62 mg, 0.517 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC.HCl, 98.8 mg, 0.517 mmol) in anhydrous dichloromethane (4 ml), after stirring for 10 min in an ice bath, adding diisopropylethylamine (0.115 ml, 1.21 mmol), continue stirring for 30 min in an ice bath, and slowly adding dichloromethane solution (4 ml) dissolved with 3,4-trans-1-benzyl-4-phenyl-3-amino piperidine (Intermediate 23, 82.8 mg, 0.31 mmol). Stirring overnight at room temperature overnight, monitor the reaction with TLC thin-layer chromatography for whether it is completed. After the reaction is completed, extracting the reaction liquid with dichloromethane 3 times, washing the merged organic phase with saturated sodium chloride once, drying it with anhydrous sodium sulfate, and recycling the solvent under reduced pressure. Dissolving the above recovered mixture into 1,2-dichloroethane (5 ml), slowly adding chloroethyl chloroformate (196 mg, 1.38 mmol) thereto, refluxing for 4 h, monitor the reaction with TLC thin-layer chromatography for whether it is completed, after the reaction is completed, recycling the solvent, adding 5 ml of methanol, and refluxing for 2 h. Recycling methanol, wash with saturated NaHCO3 solution once, extracting the reaction liquid with ethyl acetate twice, merging the organic phase, and drying it with anhydrous sodium sulfate. Carrying out column chromatography on silica gel (ethyl acetate:methanol:triethylamine=10:1:0.1), 32 mg of light yellow oily liquid (Compound 1) is obtained and the yield is 21.5%; 1H NMR (500 MHz, d-DMSO) δ 8.51-8.54 (d, J=11.35 Hz, 1H), 8.17 (s, 1H), 7.40 (s, 1H), 7.17-7.33 (m, 6H), 6.45 (s, 1H), 4.55 (m, 1H), 4.21 (s, 2H), 3.87 (s, 3H), 3.38 (m, 2H), 2.93-3.07 (m, 3H), 1.96 (m, 2H); ESI (M+H)+=351.
By using Intermediate 2 and Intermediate 24 as raw materials, the target product is synthesized and obtained according to the synthesis methods as in Example 41, the yield is 26.6%; 1H NMR (500 MHz, d-DMSO) δ 8.75-8.77 (d, J=10.90 Hz, 1H), 7.68 (s, 1H), 7.46 (s, 1H), 7.35-7.37 (d, J=10.25 Hz, 2H), 7.28-7.30 (d, J=10.25 Hz, 2H), 4.54 (m, 1H), 4.22 (s, 2H), 3.74 (s, 3H), 3.39 (m, 2H), 3.08 (m, 2H), 2.96 (m, 1H), 1.96 (m, 2H); ESI (M+H)+=453.
By using Intermediate 16 and Intermediate 25 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 29.1%; ESI (M+H)+=498.
By using Intermediate 17 and Intermediate 26 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 35.5%; ESI (M+H)+=539.
By using Intermediate 8 and Intermediate 27 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 29.2%; ESI (M+H)+=451.
By using Intermediate 11 and Intermediate 32 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 33.1%; ESI (M+H)+=443.
By using Intermediate 19 and Intermediate 28 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 34.1%; ESI (M+H)+=400.
By using Intermediate 20 and Intermediate 31 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 38.2%; ESI (M+H)+=464.
By using Intermediate 9 and Intermediate 33 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 36.8%; ESI (M+H)+=488.
By using Intermediate 11 and Intermediate 29 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 33.6%; ESI (M+H)+=467.
By using Intermediate 19 and Intermediate 30 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 37.2%; ESI (M+H)+=446.
By using Intermediate 21 and Intermediate 35 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 28.4%; ESI (M+H)+=452.
By using Intermediate 20 and Intermediate 36 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 35.1%; ESI (M+H)+=405.
By using Intermediate 9 and Intermediate 34 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 32.9%; ESI (M+H)+=506.
By using Intermediate 12 and Intermediate 37 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 39.0%; ESI (M+H)+=436.
By using Intermediate 13 and Intermediate 38 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 28.1%; ESI (M+H)+=540.
By using Intermediate 14 and Intermediate 39 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 27.6%; ESI (M+H)+=427.
By using Intermediate 18 and Intermediate 40 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 24.1%; ESI (M+H)+=456.
By using Intermediate 15 and Intermediate 23 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 34.7%; ESI (M+H)+=352.
By using Intermediate 10 and Intermediate 27 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 26.9%; ESI (M+H)+=452.
By using Intermediate 1 and Intermediate 27 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 31.6%; 1H NMR (500 MHz, d-DMSO) δ 8.61 (d, J=10.75 Hz, 1H), 8.20 (s, 1H), 7.92 (d, J=15.75 Hz, 1H), 7.21-7.41 (m, 3H), 7.11 (m, 1H), 6.48 (s, 1H), 4.49 (m, 1H), 4.18 (s, 2H), 3.88 (s, 3H), 3.35 (m, 2H), 3.08 (m, 1H), 2.94 (m, 1H), 2.85 (m, 1H), 1.97 (m, 2H); ESI (M+H)+=387.
By using Intermediate 4 and Intermediate 23 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 26.5%; 1H NMR (500 MHz, d-DMSO) δ 8.54-8.56 (d, J=8.55 Hz, 1H), 8.22 (s, 1H), 7.62 (s, 1H), 7.34 (s, 1H), 7.16-7.30 (m, 6H), 4.50 (m, 1H), 4.05 (s, 2H), 3.82 (s, 3H), 3.34 (m, 2H), 3.04 (m, 1H), 2.94 (m, 1H), 2.85 (m, 1H), 1.98 (m, 1H), 1.86 (m, 1H); ESI (M+H)+=429.
By using Intermediate 3 and Intermediate 24 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 29.5%; 1H NMR (500 MHz, d-DMSO) δ 8.62-8.64 (d, J=10.15 Hz, 1H), 8.25 (s, 1H), 7.62 (s, 1H), 7.25-7.38 (m, 5H), 4.50 (m, 1H), 4.22 (s, 2H), 3.84 (s, 3H), 3.36 (m, 2H), 2.91 (m, 3H), 1.95 (m, 2H); ESI (M+H)+=419.
By using Intermediate 3 and Intermediate 25 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 28.1%; 1H NMR (500 MHz, d-DMSO) δ 8.71 (d, 1H), 8.22 (s, 1H), 7.67 (m, 2H), 7.60 (s, 1H), 7.51 (m, 2H), 7.39 (s, 1H), 4.59 (m, 1H), 4.19 (s, 2H), 3.82 (s, 3H), 3.38 (m, 2H), 3.23 (m, 1H), 2.96 (m, 2H), 1.99 (m, 2H); ESI (M+H)+=453.
By using Intermediate 3 and Intermediate 27 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 26.7%; 1H NMR (500 MHz, d-DMSO) δ 8.70-8.72 (d, J=11.05 Hz, 1H), 8.23 (s, 1H), 7.59 (s, 1H), 7.41 (s, 1H), 7.12-7.31 (m, 1H), 4.50 (m, 1H), 4.28 (s, 2H), 3.83 (s, 3H), 3.39 (m, 2H), 3.07 (m, 2H), 2.98 (m, 1H), 1.99 (m, 2H); ESI (M+H)+=421.
By using Intermediate 4 and Intermediate 24 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 22.2%; 1H NMR (500 MHz, d-DMSO) δ 8.65-8.67 (d, J=11.15 Hz, 1H), 8.23 (s, 1H), 7.62 (s, 1H), 7.38 (s, 1H), 7.35-7.37 (d, J=10.25 Hz, 2H), 7.28-7.31 (d, J=10.25 Hz, 2H), 4.52 (m, 1H), 4.22 (s, 2H), 3.84 (s, 3H), 3.39 (m, 2H), 3.10 (m, 1H), 2.95 (m, 2H), 1.97 (m, 2H); ESI (M+H)+=463.
By using Intermediate 4 and Intermediate 25 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 26.8%; 1H NMR (500 MHz, d-DMSO) δ 8.77-8.79 (d, J=10.95 Hz, 1H), 8.23 (s, 1H), 7.67-7.68 (d, J=5.4 Hz, 2H), 7.62 (s, 1H), 7.52 (d, J=5.4 Hz, 2H), 7.39 (s, 1H), 4.60 (m, 1H), 4.19 (s, 4H), 3.83 (s, 3H), 3.39 (m, 2H), 3.23 (m, 1H), 2.96 (m, 2H), 2.00 (m, 2H); ESI (M+H)+=497.
By using Intermediate 4 and Intermediate 27 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 22.5%; 1H NMR (500 MHz, d-DMSO) δ 8.62-8.64 (d, J=10.90 Hz, 1H), 8.25 (s, 1H), 7.62 (s, 1H), 7.38 (s, 1H), 7.08-7.36 (m, 3H), 4.44 (m, 1H), 4.19 (s, 2H), 3.83 (s, 3H), 3.37 (m, 2H), 3.30 (m, 1H), 2.91 (m, 2H), 1.98 (m, 1H), 1.90 (m, 1H); ESI (M+H)+=465.
By using Intermediate 2 and Intermediate 23 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 28.6%; 1H NMR (500 MHz, d-DMSO) δ 8.77-8.79 (d, J=11.15 Hz, 1H), 7.71 (s, 1H), 7.46 (s, 1H), 7.24-7.33 (m, 5H), 4.59 (m, 1H), 4.26 (s, 2H), 3.77 (s, 3H), 3.42 (m, 2H), 3.12 (m, 1H), 3.00 (m, 2H), 2.01 (m, 2H); ESI (M+H)+=419.
By using Intermediate 2 and Intermediate 25 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 29.9%; 1H NMR (500 MHz, d-DMSO) δ 8.79-8.81 (d, J=10.85 Hz, 1H), 7.68 (s, 1H), 7.49 (s, 1H), 7.13-7.38 (m, 4H), 4.49 (m, 1H), 4.21 (s, 4H), 3.74 (s, 3H), 3.36 (m, 2H), 3.10 (m, 1H), 2.95 (m, 2H), 1.98 (m, 2H); ESI (M+H)+=487.
By using Intermediate 2 and Intermediate 25 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 33.9%; 1H NMR (500 MHz, d-DMSO) δ 8.63-8.65 (d, J=9.05 Hz, 1H), 8.23 (s, 1H), 7.62 (s, 1H), 7.36 (s, 1H), 7.10-7.34 (m, 3H), 4.46 (m, 1H), 4.21 (s, 2H), 3.82 (s, 3H), 3.36 (m, 3H), 2.90 (m, 2H), 2.85 (m, 1H), 1.98 (m, 1H), 1.91 (m, 1H); ESI (M+H)+=455.
By using Intermediate 5 and Intermediate 24 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 31.2%; 1H NMR (500 MHz, d-DMSO) δ 8.70-8.72 (d, J=10.55 Hz, 1H), 7.95 (s, 1H), 7.90 (s, 1H), 7.64 (s, 1H), 7.43 (s, 1H), 7.30-7.34 (m, 4H), 4.48 (m, 1H), 4.25 (s, 2H), 3.89 (s, 3H), 3.55 (m, 1H), 3.39 (m, 1H), 3.00 (m, 3H), 1.97 (m, 2H). ESI (M+H)+=401.
By using Intermediate 5 and Intermediate 27 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 32.5%; 1H NMR (500 MHz, d-DMSO) δ 8.78-8.80 (d, J=10.85 (Hz, 1H), 7.96 (s, 1H), 7.92 (s, 1H), 7.44 (s, 1H), 7.14-7.36 (m, 3H), 6.43 (s, 1H), 4.45 (m, 1H), 4.24 (s, 2H), 3.90 (s, 3H), 3.39 (m, 2H), 3.00 (m, 1H), 3.09 (m, 2H), 2.01 (m, 2H); ESI (M+H)+=403.
By using Intermediate 6 and Intermediate 23 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 23.8%; 1H NMR (500 MHz, d-DMSO) δ 8.64-8.66 (d, J=10.65 Hz, 1H), 7.96 (s, 1H), 7.81 (s, 1H), 7.56 (s, 1H), 7.11-7.21 (m, 5H), 4.42 (m, 1H), 4.11 (s, 2H), 3.74 (s, 3H), 3.32 (m, 2H), 2.85-2.96 (m, 3H), 1.92 (m, 2H); ESI (M+H)+=401.
By using Intermediate 6 and Intermediate 24 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 28.6%; 1H NMR (500 MHz, d-DMSO) δ 8.76-8.78 (d, J=9.80 Hz, 1H), 8.04 (s, 1H), 7.89 (s, 1H), 7.64 (s, 1H), 7.33 (m, 4H), 4.46 (m, 1H), 4.19 (s, 2H), 3.82 (s, 3H), 3.39 (m, 2H), 2.97-3.08 (m, 3H), 1.98 (m, 2H); ESI (M+H)+=435.
By using Intermediate 6 and Intermediate 27 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 25.1%; 1H NMR (500 MHz, d-DMSO) δ 8.79-8.81 (d, J=10.10 (Hz, 1H), 8.06 (s, 1H), 7.92 (s, 1H), 7.64 (s, 1H), 7.15-7.36 (m, 3H), 4.47 (m, 1H), 4.24 (s, 2H), 3.84 (s, 3H), 3.42 (m, 2H), 3.09 (m, 2H), 3.01 (m, 1H), 2.03 (m, 2H); ESI (M+H)+=437.
By using Intermediate 7 and Intermediate 24 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 27.0%; 1H NMR (500 MHz, d-DMSO) δ 8.66 (d, 1H), 8.04 (s, 1H), 7.83 (s, 1H), 7.64 (s, 1H), 7.34-7.36 (d, J=10.25 Hz, 2H), 7.27-7.29 (d, J=10.25 Hz, 2H), 4.43 (m, 1H), 4.22 (s, 2H), 3.80 (s, 3H), 3.40 (m, 2H), 3.08 (m, 2H), 2.89 (m, 1H), 1.97 (m, 2H); ESI (M+H)+=479.
By using Intermediate 7 and Intermediate 24 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 35.3%; 1H NMR (500 MHz, d-DMSO) δ 8.82-8.84 (d, J=10.25 Hz, 1H), 8.05 (s, 1H), 7.91 (s, 1H), 7.65 (s, 1H), 7.16-7.36 (m, 3H), 4.51 (m, 1H), 4.31 (s, 2H), 3.84 (s, 3H), 3.45 (m, 2H), 3.01 (m, 3H), 2.05 (m, 2H); ESI (M+H)+=481.
By using Intermediate 2 and Intermediate 59 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 32.1%; 1H NMR (500 MHz, DMSO) δ 8.79 (d, J=8.0 Hz, 1H), 7.66 (s, 1H), 7.54 (t, J=8.7 Hz, 1H), 7.49 (d, J=9.0 Hz, 1H), 7.46 (s, 1H), 7.26 (dd, J=18.3, 8.5 Hz, 1H), 4.47 (d, J=7.2 Hz, 1H), 4.10 (s, 2H), 3.71 (s, 3H), 3.35 (dd, J=13.9, 6.9 Hz, 2H), 3.13-3.00 (m, 1H), 2.90 (s, 2H), 1.91 (d, J=28.3 Hz, 2H). ESI (M+H)+=488.
By using Intermediate 2 and Intermediate 56 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 33.5%; ESI (M+H)+=488.
By using Intermediate 7 and Intermediate 60 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 37.9%; ESI (M+H)+=481.
By using Intermediate 7 and Intermediate 57 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 35.7%; ESI (M+H)+=481.
By using Intermediate 2 and Intermediate 61 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 39.2%; 1H NMR (500 MHz, d-DMSO) δ 8.79-8.81 (d, J=10.85 Hz, 1H), 7.68 (s, 1H), 7.49 (s, 1H), 7.13-7.38 (m, 4H), 4.49 (m, 1H), 4.21 (s, 4H), 3.74 (s, 3H), 3.36 (m, 2H), 3.10 (m, 1H), 2.95 (m, 2H), 1.98 (m, 2H). ESI (M+H)+=487.
By using Intermediate 2 and Intermediate 58 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 28.4%; ESI (M+H)+=487.
By using Intermediate 41 and Intermediate 23 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 38.3%; ESI (M+H)+=401.
By using Intermediate 42 and Intermediate 27 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 39.1%; ESI (M+H)+=430.
By using Intermediate 43 and Intermediate 24 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 34.9%; ESI (M+H)+=439.
By using Intermediate 44 and Intermediate 32 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 37.4%; ESI (M+H)+=442.
By using Intermediate 45 and Intermediate 25 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 39.2%; ESI (M+H)+=536.
By using Intermediate 46 and Intermediate 26 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 32.6%; ESI (M+H)+=537.
By using Intermediate 47 and Intermediate 37 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 45.1%; ESI (M+H)+=449.
By using Intermediate 48 and Intermediate 27 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 35.7%; ESI (M+H)+=447.
By using Intermediate 49 and Intermediate 24 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 39.4%; ESI (M+H)+=457.
By using Intermediate 50 and Intermediate 30 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 37.3%; ESI (M+H)+=541.
By using Intermediate 51 and Intermediate 38 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 42.5%; ESI (M+H)+=451.
By using Intermediate 52 and Intermediate 37 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 48.1%; ESI (M+H)+=450.
By using Intermediate 53 and Intermediate 28 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 44.5%; ESI (M+H)+=481.
By using Intermediate 54 and Intermediate 32 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 47.3%; ESI (M+H)+=455.
By using Intermediate 54 and Intermediate 39 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 39.7%; ESI (M+H)+=461.
By using Intermediate 55 and Intermediate 35 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 32.9%; ESI (M+H)+=380.
By using Intermediate 42 and Intermediate 60 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 35.1%; ESI (M+H)+=430.
By using Intermediate 42 and Intermediate 57 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 31.2%; ESI (M+H)+=430.
By using Intermediate 45 and Intermediate 61 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 33.9%; ESI (M+H)+=537.
By using Intermediate 45 and Intermediate 58 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 38.2%; ESI (M+H)+=537.
By using Intermediate 1 and Intermediate 63 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 23.4%; 1H NMR (500 MHz, CDCl3) δ 7.61 (d, J=0.9 Hz, 1H), 7.47 (d, J=1.9 Hz, 1H), 7.38-7.40 (d, J=7.75 z, 1H), 7.30 (d, J=0.9 Hz, 1H), 7.09-7.20 (m, 3H), 6.32 (d, J=1.9 Hz, 1H), 4.66 (m, 1H), 3.92 (s, 3H), 3.62 (m, 1H), 3.52 (m, 1H), 3.44 (m, 1H), 3.18 (m, 2H). ESI (M+H)+=373.
By using Intermediate 3 and Intermediate 63 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 33.5%; 1H NMR (500 MHz, CDCl3) δ 7.75 (s, 1H), 7.57-7.59 (d, J=7.65 z, 1H), 7.43 (s, 1H), 7.39 (s, 1H), 7.07-7.20 (m, 3H), 4.69 (m, 1H), 3.86 (s, 3H), 3.68 (m, 1H), 3.59 (m, 1H), 3.50 (m, 1H), 3.26 (s, 1H) 3.20 (m, 1H). ESI (M+H)+=407.
By using Intermediate 4 and Intermediate 62 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 28.2%; 1HNMR (500 MHz, d-DMSO) δ 8.91 (d, J=8.2 Hz, 1H), 8.29 (s, 1H), 7.70-7.72 (d, J=8.1 Hz, 2H), 7.65 (s, 1H), 7.60-7.62 (d, J=8.1 Hz, 2H), 7.47 (s, 1H), 4.65 (m, 1H), 3.86 (s, 3H), 3.59 (m, 2H), 3.49 (m, 1H), 3.11 (m, 1H), 3.02 (m, 1H). ESI (M+H)+=448.
By using Intermediate 4 and Intermediate 63 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 36.7%; 1H NMR (500 MHz, CDCl3) δ 7.78 (s, 1H), 7.73-7.74 (d, J=6.75 z, 1H), 7.48 (s, 1H), 7.41 (s, 1H), 7.10-7.24 (m, 3H), 4.72 (m, 1H), 3.88 (s, 3H), 3.72 (m, 1H), 3.58 (m, 2H), 3.26-3.30 (m, 2H). ESI (M+H)+=415.
By using Intermediate 2 and Intermediate 62 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 39.5%; 1H NMR (500 MHz, d-DMSO) δ 9.03 (d, J=8.1 Hz, 1H), 7.71-7.72 (d, J=8.1 Hz, 2H), 7.69 (s, 1H), 7.61-7.63 (d, J=8.1 Hz, 2H), 7.53 (s, 1H), 4.71 (m, 1H), 3.74 (s, 3H), 3.65 (m, 2H), 3.52 (m, 1H), 3.19 (m, 1H), 3.09 (m, 1H). ESI (M+H)+=448.
By using Intermediate 42 and Intermediate 63 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 40.5%; ESI (M+H)+=473.
By using Intermediate 42 and Intermediate 62 as raw materials, the target product is prepared and obtained according to the methods as in Example 41; the yield is 38.2%; ESI (M+H)+=473.
Sequentially adding 4-methyl bromobenzoate (Compound 6-1) (2.2 g, 10 mmol), tetra(triphenylphosphine)palladium (1.15 g, 1 mmol), 1-methyl-1H-pyrazol-5-boric acid pinacol ester (2.5 g, 12 mmol) and potassium phosphate trihydrate (4.0 g, 15 mmol) to 100 ml of three-neck flask with 50 mL of DMF under the protection of N2, fully stirring the reaction system at 90° C. and reacting overnight. Cooling the product to room temperature after the reaction is completed, pouring the reaction liquid into 100 ml of water, extracting the reaction liquid with ethyl acetate 3 times, merging the organic layer, washing with saturated sodium chloride twice, drying it with anhydrous sodium sulfate, concentrating under reduced pressure, and purify the obtained primary product by column chromatography on silica gel, 1.83 g of light yellow solid (Intermediate 6-2) is obtained and the yield is 85%; 1H NMR (500 MHz, CDCl3) δ 8.12 (d, J=8.3 Hz, 2H), 7.54 (d, J=1.9 Hz, 1H), 7.51 (d, J=8.3 Hz, 2H), 6.38 (d, J=1.9 Hz, 1H), 3.95 (s, 3H), 3.93 (s, 3H).
Dissolving Intermediate 6-2 (1.1 g, 5 mmol) in 20 ml tetrahydrofuran, slowly adding NBS (1.1 g, 6 mmol), after reacting for 5 h at room temperature, adding 10 ml of 6N NaOH aqueous solution, continue reacting for 6 h at room temperature, and removing the organic solvent under reduced pressure. Adding 10 ml of water to the remaining reaction mixture, washing with dichloromethane twice, adjusting the water layer with 1N HCl solution to pH of about 3, a large amount of solid and filter is precipitated, washing the filter cake once and drying it, 1.2 g of white solid (Intermediate 6-4) is obtained and the yield is 86%; 1H NMR (500 MHz, CDCl3) δ 8.26 (d, J=8.3 Hz, 2H), 7.59 (s, 1H), 7.56 (d, J=8.3 Hz, 2H), 3.87 (s, 3H).
Dissolving 2-nitroethyl tert-butyl carbamate (Compound 6-5, 380 mg, 2 mmol), ((S)-(−)-α,α-diphenyl-2-pyrrylmethyl)trimethylsilyl ether (33 mg, 0.1 mmol), benzoic acid (25 mg, 0.2 mmol) in anhydrous dichloromethane (2 ml), slowly adding 4-chlorocinnamaldehyde (Intermediate 1-6, 167 mg, 1 mmol) in an ice bath, stirring for about 24 h at room temperature, diluting the reaction system with dichloromethane to 10 ml, slowly dropwise adding 200 μl of trifluoroacetic acid to the reaction liquid, reacting for 5 h at room temperature, subsequently adding about 10 ml of 1N NaHCO3 solution to the reaction liquid, stirring for 10 min at room temperature, and then extracting the reaction liquid with ethyl acetate 3 times, washing the merged organic phase with saturated sodium chloride once, drying it with anhydrous sodium sulfate, and purifying by column chromatography, 260 mg of light yellow solid (Intermediate 6-7) is obtained and the yield is 77%; 1H NMR (500 MHz, CDCl3) δ 7.32 (d, J=8.4 Hz, 2H), 7.18 (d, J=8.4 Hz, 2H), 7.16-6.96 (m, 1H), 4.94-4.78 (m, 1H), 4.61 (s, 1H), 4.21 (s, 1H), 4.09-3.91 (m, 2H), 1.52 (s, 9H).
Dissolving Intermediate 6-7 (169 mg, 0.5 mmol) in ethyl acetate (10 ml), adding 30 mg of 10% Pd/C thereto, hydrogenating overnight at room temperature, suction filtrating after the reaction is completed, and spin drying the filtrate, 110 mg of oily liquid (Intermediate 6-8) is obtained and the yield is 71%; ESI (M+H)+=311.
Dissolving Intermediate 6-4 (97 mg, 0.345 mmol), 1-hydroxybenzotriazole (HOBt) (78.62 mg, 0.517 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDC.HCl (98.8 mg, 0.517 mmol) in anhydrous dichloromethane (4 ml), adding diisopropylethylamine (0.115 ml, 1.21 mmol) thereto, after stirring for 10 min in an ice bath, continue stirring for 15 min in an ice bath, and then slowly adding dichloromethane solution (4 ml) dissolved with Intermediate 6-8 (109 mg, 0.35 mmol), and stirring overnight at room temperature. After the reaction is completed, pouring the reaction liquid into 15 ml of water, extracting the reaction liquid with dichloromethane 3 times, merging the organic phase, washing with saturated sodium chloride twice, drying it with anhydrous sodium sulfate, and spin drying; dissolving the obtained residue to a small amount of acetate ethyl, slowly adding ethyl acetate saturated with HCl thereto in an ice bath, after reacting for 2 h at room temperature, spin dry, add saturated NaHCO3 solution, extract the reaction liquid with ethyl acetate twice, merging the organic phase, drying it with anhydrous sodium sulfate, and purifying by column chromatography on silica gel, 80 mg of white powder (Compound 72) is obtained and the yield is 49%; 1H NMR (500 MHz, CDCl3) δ 7.63 (d, J=8.4 Hz, 2H), 7.49 (s, 1H), 7.40 (d, J=8.4 Hz, 2H), 7.28 (d, J=8.5 Hz, 2H), 7.24 (d, J=8.5 Hz, 2H), 5.94 (d, J=6.5 Hz, 1H), 4.32 (m, 1H), 3.76 (s, 3H), 3.59 (m, 1H), 3.20 (d, J=12.7 Hz, 1H), 2.81-2.70 (m, 2H), 2.66-2.57 (m, 1H), 2.25-2.15 (m, 1H), 1.82; ESI (M+H)+=473.
By using Intermediate 6-2 and 3-fluorocinnamaldehyde as raw materials, Compound 73 is prepared and obtained according to the methods as in Example 112, the yield is 34%; 1H NMR (500 MHz, MeOD) δ 7.77 (d, J=8.4 Hz, 2H), 7.58 (s, 1H), 7.55-7.51 (m, 2H), 7.35 (td, J=8.0, 6.1 Hz, 1H), 7.21 (d, J=7.8 Hz, 1H), 7.17-7.13 (m, 1H), 6.98 (td, J=8.3, 2.0 Hz, 1H), 4.67 (td, J=11.6, 4.4 Hz, 1H), 3.79 (s, 3H), 3.69-3.64 (m, 1H), 3.57 (d, J=12.8 Hz, 1H), 3.29-3.19 (m, 2H), 3.16 (t, J=12.0 Hz, 1H), 2.23 (dd, J=14.5, 2.3 Hz, 1H), 2.16-2.08 (m, 1H). ESI (M+H)+=413.
By using 4-bromo-3-methyl methylbenzoate and 3-trifluoromethyl-4-chlorocinnamaldehyde as raw materials, Compound 74 is prepared and obtained according to the methods as in Example 112, the yield is 39%; ESI (M+H)+=511.
By using 2,5-difluoro-4-bromo-methyl benzoate and 3-trifluoromethyl-4-chlorocinnamaldehyde as raw materials, Compound 75 is prepared and obtained according to the methods as in Example 112, the yield is 29%; ESI (M+H)+=527.
By using 3-cyano-4-bromo-methyl benzoate and 4-chlorocinnamaldehyde as raw materials, Compound 76 is prepared and obtained according to the methods as in Example 112, the yield is 21%; ESI (M+H)+=454.
By using Intermediate 6-2 and 3,4-difluorocinnamaldehyde as raw materials, Compound 77 is prepared and obtained according to the methods as in Example 112, the yield is 30%; 1H NMR (400 MHz, DMSO) δ 8.60 (d, J=8.8 Hz, 1H), 7.75 (d, J=8.3 Hz, 2H), 7.58 (d, J=8.3 Hz, 2H), 7.47 (d, J=1.9 Hz, 1H), 7.33 (tt, J=17.0, 8.4 Hz, 2H), 7.14 (s, 1H), 6.44 (d, J=1.9 Hz, 1H), 4.49 (m, 1H), 3.84 (s, 3H), 3.38 (dd, J=14.0, 7.0 Hz, 2H), 3.07 (dt, J=13.7, 8.2 Hz, 1H), 2.92 (dd, J=23.6, 12.0 Hz, 2H), 1.98 (s, 2H). ESI (M+H)+=397.
By using 3-chloro-4-bromo-methyl benzoate and 3,4-difluorocinnamaldehyde as raw materials, Compound 78 is prepared and obtained according to the methods as in Example 112, the yield is 39%; ESI (M+H)+=465.
By using 3-amido-4-bromo-methyl benzoate and 3-fluorocinnamaldehyde as raw materials, Compound 79 is prepared and obtained according to the methods as in Example 112, the yield is 32%; ESI (M+H)+=428.
By using 2-trifluoromethyl-4-bromo-methyl benzoate and 4-chlorocinnamaldehyde as raw materials, Compound 80 is prepared and obtained according to the methods as in Example 112, the yield is 28%; ESI (M+H)+=497.
By using Intermediate 6-2 and 3,4,5-trifluorocinnamaldehyde as raw materials, Compound 81 is prepared and obtained according to the methods as in Example 112, the yield is 37%; ESI (M+H)+=493.
By using 3-fluoro-4-bromo-methyl benzoate and 3,4-difluorocinnamaldehyde as raw materials, Compound 82 is prepared and obtained according to the methods as in Example 112, the yield is 33%; ESI (M+H)+=449.
By using 3-methoxyl-4-bromo-methyl benzoate and 3-fluorocinnamaldehyde as raw materials, Compound 83 is prepared and obtained according to the methods as in Example 112, the yield is 33%; ESI (M+H)+=443.
By using 2-fluoro-4-bromo-methyl benzoate and 3-trifluoromethyl-4-chlorocinnamaldehyde as raw materials, Compound 84 is prepared and obtained according to the methods as in Example 112, the yield is 27%; ESI (M+H)+=515.
By using 3-trifluoromethoxy-4-bromo-methyl benzoate and 3-fluorocinnamaldehyde as raw materials, Compound 85 is prepared and obtained according to the methods as in Example 112, the yield is 34%; ESI (M+H)+=497.
By using 3-chloro-4-bromo-methyl benzoate and 3-fluorocinnamaldehyde as raw materials, Compound 86 is prepared and obtained according to the methods as in Example 112, the yield is 31%; ESI (M+H)+=447.
By using 3-(furan-3-yl)-4-bromo-methyl benzoate and 3,4-difluorocinnamaldehyde as raw materials, Compound 87 is prepared and obtained according to the methods as in Example 112, the yield is 26%; ESI (M+H)+=497.
By using 2-(2-methylfuran-3-yl)-4-bromo-methyl benzoate and 3-fluorocinnamaldehyde as raw materials, Compound 88 is prepared and obtained according to the methods as in Example 112, the yield is 33%; ESI (M+H)+=493.
By using 3-chloro-4-bromo-methyl benzoate and (E)-3-(1H-indol-3-yl)acrylic aldehyde as raw materials, Compound 89 is prepared and obtained according to the methods as in Example 112, the yield is 35%; ESI (M+H)+=468.
By using 2-(5-chlorothiophene-3-yl)-4-bromo-methyl benzoate and 3,4-difluorocinnamaldehyde as raw materials, Compound 90 is prepared and obtained according to the methods as in Example 112, the yield is 32%; ESI (M+H)+=547.
By using 3-chloro-4-bromo-methyl benzoate and (E)-3-(1H-indol-4-yl)acrylic aldehyde as raw materials, Compound 91 is prepared and obtained according to the methods as in Example 112, the yield is 31%; ESI (M+H)+=468.
By using 5-bromo-4-chloropyridinyl-2-formic acid and 3-fluorocinnamaldehyde as raw materials, Compound 92 is prepared and obtained according to the methods as in Example 112, the yield is 40%; ESI (M+H)+=448.
By using 5-bromopyrimidine-2-formic acid and 3,4-difluorocinnamaldehyde as raw materials, Compound 93 is prepared and obtained according to the methods as in Example 112, the yield is 35%; ESI (M+H)+=433.
By using 4-methyl-5-bromo-pyridinyl-2-formic acid and 3,4-difluorocinnamaldehyde as raw materials, Compound 94 is prepared and obtained according to the methods as in Example 112, the yield is 32%; ESI (M+H)+=446.
By using 4-chloro-5-bromo-pyridinyl-2-formic acid and 3-trifluoromethyl-4-chlorocinnamaldehyde as raw materials, Compound 95 is prepared and obtained according to the methods as in Example 112, the yield is 33%; ESI (M+H)+=532.
By using 5-bromo-pyridinyl-2-formic acid and 3-trifluoromethyl-4-chlorocinnamaldehyde as raw materials, Compound 96 is prepared and obtained according to the methods as in Example 112, the yield is 43%; ESI (M+H)+=498.
By using 3-fluoro-5-bromo-pyridinyl-2-formic acid and 3,4-difluorocinnamaldehyde as raw materials, Compound 97 is prepared and obtained according to the methods as in Example 112, the yield is 39%; ESI (M+H)+=450.
Dissolving 2-nitroethyl tert-butyl carbamate (Intermediate 6-5, 2.85 g, 15 mmol), ((S)-(−)-α,α-diphenyl-2-pyrrylmethyl)trimethylsilyl ether (0.36 g, 1.1 mmol), and benzoic acid (0.25 g, 2 mmol) in anhydrous dichloromethane (15 ml), slowly adding 3,4-difluorocinnamaldehyde (Compound 4-1b, 1.68 g, 10 mmol) thereto in an ice bath, stirring for about 18 h at room temperature, diluting the reaction system with dichloromethane to 100 ml. Decreasing the temperature of the reaction system to −78° C., adding allyltrimethylsilane (5 ml, 30 mmol) into the reaction liquid, subsequently, slowly dropwise adding 2.5 ml of aether boron trifluoride, continue reacting for 10 h, adding about 100 ml of 1N NaHCO3 solution into the reaction liquid, stirring for 10 min at room temperature, and then extracting the reaction liquid with ethyl acetate 3 times, merging the organic phase, washing with saturated sodium chloride twice, drying it with anhydrous sodium sulfate, and purifying by column chromatography on silica gel, 1.9 g of white solid (Intermediate 7-2) is obtained and the yield is 50%; 1H NMR (500 MHz, CDCl3) δ 7.15-7.07 (m, 1H), 7.06-6.98 (m, 1H), 6.92 (d, J=8.3 Hz, 1H), 5.74 (s, 1H), 5.17 (d, J=16.3 Hz, 1H), 5.10 (d, J=9.8 Hz, 1H), 4.67-4.34 (m, 3H), 3.51-3.41 (m, 1H), 3.29 (dt, J=49.6, 12.0 Hz, 1H), 2.58 (s, 1H), 2.40 (d, J=19.3 Hz, 1H), 1.97-1.82 (m, 2H), 1.48 (s, 9H).
Dissolving Intermediate 7-2 (1.9 g, 5 mmol) in 40 ml solvent of mixed DCM/CH3CN/H2O (v/v/v=1/1/2), sequentially slowly adding NaIO4 (5.35 g, 25 mmol) and RuCl3 monohydrate (170 mg, 1 mmol) thereto in an ice bath, stirring overnight at room temperature, filtering to remove black insoluble substance, adjusting the filtrate with dilute hydrochloric acid solution to a pH of 5, extracting the reaction liquid with dichloromethane 3 times, merging the organic layer, washing with saturated sodium chloride twice, drying it with anhydrous sodium sulfate, and spin drying, 1.8 g of colorless oily matter (Intermediate 7-3) is obtained and the yield is 90%; 1H NMR (500 MHz, CDCl3) δ 7.11 (dd, J=18.2, 8.4 Hz, 1H), 7.05-7.00 (m, 1H), 6.92 (dd, J=5.4, 3.1 Hz, 1H), 4.89 (m, 1H), 4.62 (m, 2H), 3.42 (m, 1H), 3.29 (m, 1H), 2.76 (d, J=6.7 Hz, 2H), 1.97 (s, 2H), 1.46 (s, 9H).
Dissolving Intermediate 7-3 (200 mg, 0.5 mmol) in 5 ml of DMF, sequentially adding HBTU (379 mg, 1 mmol) and 0.25 ml of triethylamine thereto in an ice bath, dropwise adding ethanol solution (1 ml) with 30% methylamine to the reaction liquid after reacting for 15 min at room temperature, continue reacting for 3 h. After the reaction is finished, pouring the reaction system to 10 ml of water, extracting the reaction liquid with ethyl acetate 3 times, merging the organic phase, washing it with saturated sodium chloride twice, drying it with anhydrous sodium sulfate, and spin drying, 200 mg of white solid (Intermediate 7-4) is obtained and the yield is 97%, which is directly used for the next reaction without purification.
Dissolving Intermediate 7-4 (200 mg, 0.5 mmol) in ethyl acetate (10 ml), adding 30 mg of 10% Pd/C, hydrogenating overnight at room temperature, suction filtrating after the reaction is completed, and spin drying the filtrate, 180 mg of oily liquid (Intermediate 7-5) is obtained and the yield is 91%; ESI (M+H)+=384.
Dissolving Intermediate 6-2 (1.1 g, 5 mmol) in 20 ml of tetrahydrofuran, slowly adding NCS (0.8 g, 6 mmol), and adding 10 ml of 6N NaOH aqueous solution after reacting for 5 h at room temperature, continue reacting for 6 h at room temperature, and removing the organic solvent under the reduced pressure. Adding 10 ml of water to the remained reaction mixture, washing with dichloromethane twice, adjusting the water layer with 1N HCl solution to a pH of about 3, a large amount of solid is precipitedng and filtered, washing the filter cake once and drying it, 0.88 g of white solid (Intermediate 7-6) is obtained and the yield is 75%; 1H NMR (500 MHz, DMSO) δ 8.11-8.08 (m, 2H), 7.70 (s, 1H), 7.68-7.64 (m, 2H), 3.80 (s, 3H).
Dissolving Intermediate 7-6 (81 mg, 0.345 mmol), 1-hydroxybenzotriazole (HOBt) (78.62 mg, 0.517 mmol) and 1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride EDC.HCl (98.8 mg, 0.517 mmol) in anhydrous dichloromethane (4 ml), adding diisopropylethylamine (0.115 ml, 1.21 mmol) thereto after stirring for 10 min in an ice bath, continue stirring for 15 min in an ice bath, and then slowly adding dichloromethane solution (4 ml) dissolved with Intermediate 7-5 (134 mg, 0.35 mmol), and stirring overnight at room temperature. After the reaction is completed, pouring the reaction liquid to 15 ml of water, extracting the reaction liquid with dichloromethane 3 times, merging the organic phase, washing it with saturated sodium chloride twice, drying it with anhydrous sodium sulfate, and spin drying; dissolving the obtained residue to a small amount of acetate ethyl, slowly adding ethyl acetate saturated with HCl thereto in an ice bath, spin drying after reacting for 3 h at room temperature, adding saturated NaHCO3 solution, extracting the reaction liquid with ethyl acetate twice, merging the organic phase, drying it with anhydrous sodium sulfate, and purifying by column chromatography on silica gel, 97 mg of white powder (Compound 98) is obtained and the yield is 56%; 1H NMR (500 MHz, MeOD) δ 7.88-7.79 (m, 2H), 7.59-7.54 (m, 3H), 7.33 (ddd, J=11.6, 7.6, 1.9 Hz, 1H), 7.22 (ddd, J=15.6, 13.7, 8.5 Hz, 2H), 4.57 (td, J=11.5, 4.9 Hz, 1H), 4.16-4.08 (m, 1H), 3.80 (s, 3H), 3.54-3.47 (m, 1H), 3.46-3.37 (m, 2H), 3.08 (dd, J=16.3, 10.0 Hz, 1H), 2.81 (d, J=4.9 Hz, 3H), 2.77 (dd, J=16.3, 5.1 Hz, 1H), 2.31-2.22 (m, 1H), 2.13-2.06 (m, 1H). ESI (M+H)+=502.
By using 3-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)benzoic acid and Intermediate 7-5 as raw materials, Compound 99 is prepared and obtained according to the methods as in Example 138, the yield is 44%; ESI (M+H)+=536.
By using Intermediate 7-6 and Intermediate 7-2 as raw materials, Compound 100 is prepared and obtained by condensation, oxidation, deprotection steps and the like according to the methods as in Example 138, the yield is 47%; ESI (M+H)+=475.
By using 3-fluoro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)benzoic acid and Intermediate 7-2 as raw materials, Compound 101 is prepared and obtained according to the methods as in Example 138, the yield is 38%; ESI (M+H)+=556.
By using 3-methyl-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)benzoic acid and Intermediate 7-2 as raw materials, Compound 102 is prepared and obtained according to the methods as in Example 138, the yield is 19%; ESI (M+H)+=556.
By using 3-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)benzoic acid and Intermediate 7-2 as raw materials, Compound 103 is prepared and obtained according to the methods as in Example 138, the yield is 31%; ESI (M+H)+=568.
Dissolving Intermediate 1-7 (191 mg, 1 mmol), N-chlorosuccinimide (399 mg, 3 mmol) in DMF/THF (v/v=1:1, 10 ml), raising temperature to 80° C. and reacting for about 3 h, cooling the product to room temperature after the reaction is completed, pouring the system to water, and then extracting the reaction liquid with ethyl acetate 3 times, washing the merged organic phase with saturated sodium chloride once, and drying it with anhydrous sodium sulfate. Recycling the solvent under reduced pressure, and purifying it by column chromatography on silica gel, 160 mg of light yellow solid (Intermediate 8-1) is obtained and the yield is 61%; 1H NMR (500 MHz, CDCl3) δ 10.00 (d, J=1.1 Hz, 1H), 7.92 (t, J=1.2 Hz, 1H), 7.91 (d, J=1.4 Hz, 1H), 3.87 (s, 3H).
Dissolving Intermediate 8-1 (520 mg, 2 mmol) in acetone (5 ml), then slowly adding KMnC4 (380 mg, 2.4 mmol) thereto, stirring for 2 h at room temperature, suction filtrating after the reaction is completed, washing filter cake with ethyl acetate twice, concentrating the merged filtrate and recrystallizing thereof with ethyl acetate, 0.44 g of white solid (Intermediate 8-2) is obtained and the yield is 80%; 1H NMR (500 MHz, DMSO) δ 7.99 (s, 1H), 7.67 (s, 1H), 3.82 (s, 3H).
Dissolving Intermediate 8-2 (95 mg, 0.345 mmol), 1-hydroxybenzotriazole (HOBt) (78.62 mg, 0.517 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDC.HCl (98.8 mg, 0.517 mmol) in anhydrous dichloromethane (4 ml), adding diisopropylethylamine (0.115 ml, 1.21 mmol) after stirring for 10 min in an ice bath, after continue stirring for 15 min in an ice bath, slowly adding dichloromethane solution (4 ml) dissolved with Intermediate 7-5 (134 mg, 0.35 mmol), and stirring overnight at room temperature. After the reaction is completed, pouring the reaction liquid to 15 ml of water, extracting thereof with dichloromethane 3 times, washing the merged organic phase with saturated sodium chloride twice, drying it with anhydrous sodium sulfate, and spin drying; dissolving the obtained residue to a small amount of acetate ethyl, slowly adding HCl saturated ethyl acetate in an ice bath, spin drying after reacting for 3 h at room temperature, adding saturated NaHCO3 solution, extracting the reaction liquid with ethyl acetate twice, merging the organic phase, drying it with anhydrous sodium sulfate, and purifying by column chromatography on silica gel, 125 mg of white powder (Compound 104) is obtained and the yield is 67%; 1H NMR (500 MHz, MeOD) δ 7.99 (dd, J=4.9, 1.5 Hz, 1H), 7.88 (d, J=1.4 Hz, 1H), 7.31 (ddd, J=11.5, 7.6, 1.9 Hz, 1H), 7.25-7.15 (m, 2H), 4.50 (td, J=11.3, 5.0 Hz, 1H), 4.15-4.09 (m, 1H), 3.85 (s, 3H), 3.50-3.45 (m, 1H), 3.44-3.37 (m, 2H), 3.06 (dd, J=16.2, 9.9 Hz, 1H), 2.80 (d, J=4.8 Hz, 3H), 2.76 (dd, J=16.3, 5.2 Hz, 1H), 2.30-2.21 (m, 1H), 2.08 (dd, J=11.3, 2.0 Hz, 1H). ESI (M+H)+=542.
By using 5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-formic acid and Intermediate 7-5 as raw materials, Compound 105 is prepared and obtained according to the methods as in Example 144, the yield is 48%; 1H NMR (400 MHz, MeOD) δ 7.55 (s, 1H), 7.40-7.28 (m, 2H), 7.24-7.14 (m, 2H), 4.63-4.53 (m, 1H), 4.13 (d, J=3.7 Hz, 1H), 3.75 (d, J=12.0 Hz, 3H), 3.52-3.35 (m, 3H), 3.11-3.01 (m, 1H), 2.86-2.72 (m, 4H), 2.28 (td, J=14.7, 4.9 Hz, 1H), 2.13-2.02 (m, 1H). ESI (M+H)+=526.
By using 5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-formic acid and (4S,5S)-2-allyl-4-(3,4-difluorophenyl)-5-amidopiperidine-1-tert-butyl formate as raw materials, Compound 106 is prepared and obtained according to the methods as in Example 144, the yield is 41%; 1H NMR (500 MHz, MeOD) δ 7.53 (s, 1H), 7.51 (d, J=2.0 Hz, 1H), 7.47-7.43 (m, 1H), 7.30 (s, 1H), 7.26 (dd, J=8.3, 2.0 Hz, 1H), 5.89-5.81 (m, 1H), 5.41 (dd, J=17.0, 1.1 Hz, 1H), 5.30 (d, J=10.3 Hz, 1H), 4.57 (td, J=11.5, 5.0 Hz, 1H), 3.82-3.76 (m, 1H), 3.76 (s, 3H), 3.43-3.31 (m, 3H), 2.81-2.68 (m, 2H), 2.20-2.07 (m, 2H). ESI (M+H)+=529.
Dissolving Intermediate 70 (70 mg, 0.1 mmol), dimethylamine hydrochloride (81 mg, 1 mmol), diisopropylethylamine (0.26 ml, 1.5 mmol) in anhydrous DMF (10 ml), and reacting overnight at 55° C. under the protection of N2. Adding 30 ml of saturated sodium chloride solution to the reaction liquid for diluting, extracting thereof with ethyl acetate 3 times, merging the organic layer, drying it with anhydrous sodium sulfate, spin drying and carrying out column chromatography, 32 mg of white solid is obtained. Dissolving the obtained white solid in dichloromethane (5 ml), slowly dropwise adding trifluoroacetic acid (0.5 ml) in an ice bath, stirring for 30 min at room temperature, and recycling the solvent under reduced pressure, 18 mg of light yellow solid (Compound 107) is directly obtained and the yield is 30.5%; 1H NMR (500 MHz, DMSO) δ 9.56 (d, J=9.7 Hz, 1H), 9.01 (d, J=9.2 Hz, 1H), 7.67 (s, 1H), 7.61 (s, 1H), 7.59-7.54 (m, 2H), 7.30 (dd, J=8.4, 1.9 Hz, 1H), 4.59-4.50 (m, 1H), 3.78-3.67 (m, 4H), 3.49-3.44 (m, 1H), 3.30-3.15 (m, 4H), 2.79 (d, J=4.1 Hz, 3H), 2.76 (d, J=4.1 Hz, 3H), 2.30 (dd, J=13.7, 7.3 Hz, 2H), 2.19-2.11 (m, 1H), 1.95 (d, J=14.5 Hz, 1H). ESI (M+H)+=560.
Dissolving Intermediate 70 (70 mg, 0.1 mmol), piperidine (86 mg, 1 mmol), and diisopropylethylamine (0.26 ml, 1.5 mmol) in anhydrous DMF (10 ml), and reacting overnight at 60° C. under the protection of N2. Adding 30 ml of saturated sodium chloride solution into the reaction liquid for diluting, extracting the reaction liquid with ethyl acetate 3 times, merging the organic layer, drying it with anhydrous sodium sulfate, spin drying and carrying out column chromatography, 29 mg of white solid is obtained. Dissolve the obtained white solid in dichloromethane (5 ml), slowly dropwise adding trifluoroacetic acid (0.5 ml) thereto in an ice bath, stirring for 30 min at room temperature, and recycling the solvent under reduced pressure, 21 mg of light yellow solid (Compound 108) is directly obtained and the yield is 33.2%; 1H NMR (500 MHz, DMSO) δ 9.55 (s, 1H), 8.97 (d, J=5.5 Hz, 1H), 7.68 (s, 1H), 7.61 (d, J=2.0 Hz, 2H), 7.58 (d, J=8.3 Hz, 1H), 7.32 (dd, J=8.4, 2.0 Hz, 1H), 4.61-4.51 (m, 1H), 3.79-3.71 (m, 4H), 3.49 (d, J=11.7 Hz, 3H), 3.30-3.13 (m, 4H), 2.90 (dd, J=13.0, 8.2 Hz, 2H), 2.36 (dd, J=13.3, 7.5 Hz, 2H), 2.16 (t, J=12.6 Hz, 1H), 1.97 (d, J=11.9 Hz, 1H), 1.82 (s, 4H), 1.77-1.68 (m, 1H), 1.49-1.34 (m, 1H). ESI (M+H)+=600.
Dissolving Intermediate 70 (70 mg, 0.1 mmol), morpholine (88 mg, 1 mmol), diisopropylethylamine (0.26 ml, 1.5 mmol) in anhydrous DMF (12 ml), and reacting overnight at 60° C. under the protection of N2. Adding 30 ml of saturated sodium chloride solution into the reaction liquid for diluting, extracting thereof with ethyl acetate 3 times, merging the organic layer, drying it with anhydrous sodium sulfate, spin drying and carry out column chromatography, 30 mg of white solid is obtained. Dissolving the obtained white solid in dichloromethane (5 ml), slowly dropwise adding trifluoroacetic acid (0.5 ml) thereto in an ice bath, stirring for 30 min at room temperature, and recycling the solvent under reduced pressure, 25 mg of yellow solid (Compound 109) is obtained; the yield is 33.1%; 1H NMR (500 MHz, DMSO) δ 9.50 (s, 1H), 8.94 (s, 1H), 7.68 (s, 1H), 7.62-7.57 (m, 3H), 7.32 (dd, J=8.4, 1.9 Hz, 1H), 4.61-4.51 (m, 1H), 4.04-3.96 (m, 2H), 3.91-3.85 (m, 2H), 3.80 (s, 1H), 3.74 (s, 3H), 3.53 (d, J=12.0 Hz, 1H), 3.46-3.39 (m, 2H), 3.30 (d, J=5.8 Hz, 2H), 3.21 (dd, J=16.3, 9.6 Hz, 2H), 3.16-3.05 (m, 2H), 2.38-2.28 (m, 2H), 2.15 (t, J=13.7 Hz, 1H), 2.02-1.96 (m, 1H). ESI (M+H)+=602.
Dissolving Intermediate 70 (70 mg, 0.1 mmol), pyrrolidine (71 mg, 1 mmol), diisopropylethylamine (0.25 ml, 1.4 mmol) in anhydrous DMF (15 ml), and reacting overnight at 65° C. under the protection of N2. Adding 30 ml of saturated sodium chloride solution into the reaction liquid for diluting, extracting thereof with ethyl acetate 3 times, merging the organic layer, drying it with anhydrous sodium sulfate, spin drying and carrying out column chromatography, 26 mg of white solid is obtained. Dissolve the obtained white solid in dichloromethane (5 ml), slowly dropwise adding trifluoroacetic acid (0.5 ml) thereto in an ice bath, stirring for 30 min at room temperature, and recycling the solvent under reduced pressure, 19 mg of white solid (Compound 110) is directly obtained; the yield is 29.9%; 1H NMR (500 MHz, DMSO) δ 9.53 (d, J=10.4 Hz, 1H), 8.99 (d, J=9.2 Hz, 1H), 7.68 (s, 1H), 7.62-7.56 (m, 3H), 7.31 (dd, J=8.4, 2.0 Hz, 1H), 4.60-4.50 (m, 1H), 3.81 (s, 1H), 3.74 (s, 3H), 3.48-3.26 (m, 4H), 3.25-3.12 (m, 2H), 3.06-2.99 (m, 2H), 2.33 (t, J=12.3 Hz, 2H), 2.17 (td, J=14.4, 4.5 Hz, 1H), 2.07-1.87 (m, 6H). ESI (M+H)+=586.
Dissolving Intermediate 66 (63 mg, 0.1 mmol) in anhydrous tetrahydrofuran (2 ml), slowly dropwise adding 2N borane dimethyl sulfide complex (0.15 ml, 0.3 mmol) thereto in an ice bath, after reacting for 3 h at room temperature, dropwise adding 1 ml of 10% NaOH solution and 0.5 ml of 30% hydrogen peroxide to the reaction system at 0° C., and continue to react for 1 h. Adding Dilute 5 ml of saturated sodium chloride solution into the reaction liquid for diluting, extracting thereof with ethyl acetate 3 times, merging the organic layer, drying it with anhydrous sodium sulfate, spin drying and obtaining white solid. Dissolving this solid in dichloromethane (2 ml), slowly dropwise adding 1 ml of trifluoroacetic acid thereto, reacting for 1 h at room temperature, and recycling the solvent under reduced pressure, 22.3 mg of white solid (Compound 111) is obtained; the yield is 50%; 1H NMR (500 MHz, CDCl3) δ7.54-7.46 (m, 2H), 7.44 (s, 1H), 7.40 (d, J=8.2 Hz, 1H), 7.25-7.18 (m, 2H), 4.49-4.44 (m, 1H), 3.81-3.70 (m, 4H), 3.68-3.59 (m, 1H), 3.31-3.12 (m, 4H), 2.18-2.09 (m, 1H), 2.08-1.94 (m, 3H), 1.89-1.66 (m, 3H).
Dissolving Intermediate 67 (63 mg, 0.1 mmol) in 5 ml of dichloromethane, slowly dropwise adding 0.5 ml of trifluoroacetic acid thereto in an ice bath, reacting for 1 h under stirring at room temperature, and recycling the solvent under reduced pressure, 20.2 mg of white solid (Compound 112) is obtained; the yield is 87%; 1H NMR (500 MHz, MeOD) δ 7.53 (s, 1H), 7.52 (d, J=2.0 Hz, 1H), 7.46 (dd, J=8.3, 1.4 Hz, 1H), 7.27 (dd, J=5.9, 2.0 Hz, 2H), 4.55 (td, J=11.1, 5.0 Hz, 1H), 4.03-3.94 (m, 1H), 3.94-3.84 (m, 1H), 3.75 (s, 3H), 3.64-3.55 (m, 2H), 3.45-3.34 (m, 3H), 2.37-2.09 (m, 3H), 2.05-1.90 (m, 1H). ESI (M+H)+=561.
Dissolving in Intermediate 69 (66 mg, 0.1 mmol) in 5 ml of dichloromethane, slowly dropwise adding 1 ml of trifluoroacetic acid thereto in an ice bath, reacting for 1 h under stirring at room temperature, and recycling the solvent under reduced pressure, 29.2 mg of white solid (Compound 113) is obtained; the yield is 77%; 1H NMR (500 MHz, MeOD) δ 7.53 (s, 1H), 7.48 (dd, J=12.9, 1.9 Hz, 1H), 7.41 (t, J=7.0 Hz, 1H), 7.28-7.22 (m, 1H), 7.21 (d, J=6.2 Hz, 1H), 4.37-4.31 (m, 1H), 3.79-3.71 (m, 5H), 3.47-3.40 (m, 1H), 3.23-3.15 (m, 1H), 3.13-2.99 (m, 2H), 2.16-2.10 (m, 1H), 2.09-1.99 (m, 1H), 1.97-1.91 (m, 1H), 1.89-1.77 (m, 1H). ESI (M+H)+=531.
By using 5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-formic acid and (4S,5S)-2-(2-(4-hydroxylpiperidin-1-yl)ethyl)-4-(3,4-dichlorophenyl)-5-amidopiperidine-1-tert-butyl formate as raw materials, Compound 114 is prepared and obtained according to the methods as in Example 144, the yield is 40%; 1H NMR (500 MHz, MeOD) δ 7.62 (s, 1H), 7.53 (s, 1H), 7.45 (d, J=8.3 Hz, 1H), 7.37-7.31 (m, 2H), 4.60 (s, 1H), 4.14-3.81 (m, 2H), 3.77-3.67 (m, 4H), 3.57-3.39 (m, 5H), 3.39-3.32 (m, 2H), 3.18 (d, J=21.6 Hz, 1H), 2.62 (s, 1H), 2.40 (s, 1H), 2.26-2.09 (m, 4H), 1.97 (d, J=18.8 Hz, 1H), 1.86 (dd, J=23.7, 11.2 Hz, 1H). ESI (M+H)+=616.
By using 5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-formic acid and (4S,5S)-2-(2-(3-hydroxylpiperidin-1-yl)ethyl)-4-(3,4-dichlorophenyl)-5-amidopiperidine-1-tert-butyl formate as raw materials, Compound 115 is prepared and obtained according to the methods as in Example 144, the yield is 27%; 1H NMR (500 MHz, MeOD) δ 7.63-7.56 (m, 1H), 7.51 (s, 1H), 7.42 (d, J=8.3 Hz, 1H), 7.36-7.28 (m, 2H), 4.64-4.51 (m, 1H), 4.24-3.94 (m, 1H), 3.87-3.75 (m, 1H), 3.72 (s, 3H), 3.55-3.33 (m, 5H), 3.23-2.90 (m, 2H), 2.85-2.02 (m, 6H), 1.94-1.41 (m, 3H). ESI (M+H)+=616.
By using 5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-formic acid and 2-((4S,5S)-1-Boc-4-(3,4-dichlorophenyl)-5-amidopiperidin-2-yl)methyl acetate as raw materials, Compound 116 is prepared and obtained by amide condensation, alkaline hydrolysis, deprotection steps and the like according to the methods as in Example 144, the yield is 30%; 1H NMR (500 MHz, MeOD) δ 7.54 (s, 1H), 7.53 (d, J=1.2 Hz, 1H), 7.47-7.42 (m, 1H), 7.29 (d, J=8.2 Hz, 2H), 4.60 (d, J=6.2 Hz, 1H), 4.21-4.10 (m, 1H), 3.74 (s, 3H), 3.49-3.42 (m, 1H), 3.40-3.32 (m, 2H), 3.21-3.10 (m, 1H), 3.01-2.92 (m, 1H), 2.35-2.22 (m, 1H), 2.18-2.09 (m, 1H). ESI (M+H)+=547.
By using 5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-formic acid and (4S,5S)-2-(2-amido-2-oxoethyl)-4-(3,4-dichlorophenyl)-5-amidopiperidine-1-tert-butyl formate as raw materials, Compound 117 is prepared and obtained according to the methods as in Example 144, the yield is 40%; 1H NMR (500 MHz, MeOD) δ 7.55 (s, 1H), 7.53 (d, J=5.4 Hz, 1H), 7.46 (d, J=7.9 Hz, 1H), 7.33-7.28 (m, 2H), 4.56 (s, 1H), 4.10 (dd, J=14.2, 7.1 Hz, 1H), 3.75 (s, 3H), 3.50-3.35 (m, 3H), 3.13-3.05 (m, 1H), 2.81 (d, J=17.0 Hz, 1H), 2.26 (t, J=12.6 Hz, 1H), 2.11-2.04 (m, 1H). ESI (M+H)+=546.
By using 5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-formic acid and (4S,5S)-2-(2-methylamido-2-oxyethyl)-4-(3,4-dichlorophenyl)-5-amidopiperidine-1-tert-butyl formate as raw materials, Compound 118 is prepared and obtained according to the methods as in Example 144, the yield is 33%; 1H NMR (500 MHz, MeOD) δ 7.57 (s, 1H), 7.55 (s, 1H), 7.50-7.45 (m, 1H), 7.36-7.32 (m, 2H), 4.58 (s, 1H), 4.12 (dd, J=14.0, 6.9 Hz, 1H), 3.77 (s, 3H), 3.50-3.36 (m, 3H), 3.06 (dd, J=25.2, 15.5 Hz, 1H), 2.83-2.83 (m, 4H), 2.33-2.22 (s, 1H), 2.08 (d, J=13.3 Hz, 1H). ESI (M+H)+=560.
By using 5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-formic acid and (4S,5S)-2-(2-cyclopropylamido-2-oxoethyl)-4-(3,4-dichlorophenyl)-5-amidopiperidine-1-tert-butyl formate as raw materials, Compound 119 is prepared and obtained according to the methods as in Example 144, the yield is 32%; 1H NMR (500 MHz, MeOD) δ 7.57-7.54 (m, 2H), 7.48 (d, J=7.1 Hz, 1H), 7.37-7.29 (m, 2H), 4.58 (s, 1H), 4.17-4.10 (m, 1H), 3.77 (s, 3H), 3.50-3.35 (m, 3H), 3.03 (dd, J=17.5, 8.3 Hz, 1H), 2.79-2.71 (m, 2H), 2.27 (t, J=13.5 Hz, 1H), 2.07 (d, J=13.3 Hz, 1H), 0.82-0.72 (m, 2H), 0.63-0.52 (m, 2H). ESI (M+H)+=586.
By using 5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-formic acid and (4S,5S)-2-(2-cyclobutylamido-2-oxoethyl)-4-(3,4-dichlorophenyl)-5-amidopiperidine-1-tert-butyl formate as raw materials, Compound 120 is prepared and obtained according to the methods as in Example 144, the yield is 37%; NMR (500 MHz) δ 7.55 (s, 2H), 7.49 (d, J=8.2 Hz, 1H), 7.33 (s, 1H), 7.30 (d, J=8.3 Hz, 1H), 4.55 (s, 1H), 4.40-4.34 (m, 1H), 4.12-4.06 (m, 1H), 3.77 (s, 3H), 3.48-3.35 (m, 3H), 3.03 (dd, J=16.6, 9.5 Hz, 1H), 2.74 (d, J=14.2 Hz, 1H), 2.35-2.21 (m, 3H), 2.10-1.98 (m, 3H), 1.83-1.72 (m, 2H). ESI (M+H)+=600.
By using 5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-formic acid and (4S,5S)-2-(2-(2,3-dihydroxypropyl)amido-2-oxoethyl)-4-(3,4-dichlorophenyl)-5-amidopiperidin-1-tert-butyl formate as raw materials, Compound 121 is prepared and obtained according to the methods as in Example 144, the yield is 21%; 1H NMR (500 MHz, MeOD) δ 7.56 (s, 1H), 7.52 (d, J=1.9 Hz, 1H), 7.45 (d, J=8.3 Hz, 1H), 7.28 (dd, J=8.3, 1.9 Hz, 1H), 7.24 (s, 1H), 4.31 (td, J=10.4, 4.6 Hz, 1H), 3.77 (s, 3H), 3.75-3.71 (m, 1H), 3.63 (d, J=4.9 Hz, 1H), 3.53 (d, J=5.7 Hz, 2H), 3.50-3.37 (m, 2H), 3.30-3.24 (m, 1H), 3.11-3.04 (m, 1H), 3.03-2.97 (m, 1H), 2.83 (dd, J=14.6, 8.7 Hz, 1H), 2.57 (dd, J=14.8, 6.2 Hz, 1H), 2.10-2.03 (m, 1H), 1.94-1.88 (m, 1H). ESI (M+H)+=620.
By using 5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-formic acid and (4S,5S)-2-(2-(1H-1,2,4-triazol-1-yl)ethyl)-4-(3,4-dichlorophenyl)-5-amidopiperidine-1-tert-butyl formate as raw materials, Compound 122 is prepared and obtained according to the methods as in Example 144, the yield is 26%; 1H NMR (500 MHz, MeOD) δ 9.53 (s, 1H), 8.65 (s, 1H), 7.61 (d, J=1.9 Hz, 1H), 7.55 (s, 1H), 7.47 (d, J=8.3 Hz, 1H), 7.36 (dd, J=8.3, 1.9 Hz, 1H), 7.34 (s, 1H), 4.73 (dt, J=13.5, 6.7 Hz, 1H), 4.67-4.57 (m, 2H), 3.95 (s, 1H), 3.77 (s, 3H), 3.53-3.40 (m, 3H), 2.79 (d, J=7.2 Hz, 1H), 2.62 (dd, J=13.7, 6.4 Hz, 1H), 2.33-2.24 (m, 1H), 2.20 (d, J=13.3 Hz, 1H). ESI (M+H)+=584.
By using 5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-formic acid and (4S,5S)-2-(2-(2,3-dihydroxypropyl)amido)ethyl)-4-(3,4-dichlorophenyl)-5-amidopiperidine-1-tert-butyl formate as raw materials, Compound 123 is prepared and obtained according to the methods as in Example 144, the yield is 36%; ESI (M+H)+=606.
By using 5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-formic acid and (4S,5S)-2-(2-methoxyethyl)-4-(3,4-dichlorophenyl)-5-amidopiperidine-1-tert-butyl formate as raw materials, Compound 124 is prepared and obtained according to the methods as in Example 144, the yield is 39%; 1H NMR (500 MHz, MeOD) δ 7.54 (s, 1H), 7.51 (s, 1H), 7.47 (d, J=8.3 Hz, 1H), 7.29-7.24 (m, 2H), 4.57-4.48 (m, 1H), 3.87 (s, 1H), 3.75 (s, 3H), 3.68-3.62 (m, 2H), 3.45-3.32 (m, 6H), 2.36 (dd, J=14.1, 5.6 Hz, 1H), 2.23-2.10 (m, 2H), 2.11-2.03 (m, 1H). ESI (M+H)+=547.
By using 5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-formic acid and (4S,5S)-2-(2-((2-hydroxyethyl)amido)-2-oxoethyl)-4-(3,4-dichlorophenyl)-5-amidopiperidin-1-tert-butyl formate as raw materials, Compound 125 is prepared and obtained according to the methods as in Example 144, the yield is 21%; 1H NMR (500 MHz, MeOD) δ 7.57-7.54 (m, 2H), 7.47 (d, J=8.3 Hz, 1H), 7.31-7.28 (m, 2H), 4.48 (td, J=11.1, 4.7 Hz, 1H), 3.99-3.93 (m, 1H), 3.77 (s, 3H), 3.66 (t, J=5.7 Hz, 2H), 3.40-3.36 (m, 2H), 3.33-3.28 (m, 3H), 3.00 (dd, J=15.7, 9.5 Hz, 1H), 2.73 (dd, J=15.7, 5.5 Hz, 1H), 2.24-2.15 (m, 1H), 2.06-2.00 (m, 1H). ESI (M+H)+=590.
By using 5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-formic acid and (4S,5S)-2-(2-acetaminoethyl)-4-(3,4-difluorophenyl)-5-amidopiperidine-1-tert-butyl formate as raw materials, Compound 126 is prepared and obtained according to the methods as in Example 144, the yield is 44%; 1H NMR (500 MHz, DMSO) δ 9.67 (t, J=10.3 Hz, 1H), 9.06 (d, J=11.0 Hz, 1H), 8.80 (d, J=9.2 Hz, 1H), 8.18 (t, J=5.7 Hz, 1H), 7.68 (s, 1H), 7.53 (s, 1H), 7.38 (dd, J=19.2, 8.6 Hz, 1H), 7.31 (dd, J=9.9, 8.2 Hz, 1H), 7.12 (s, 1H), 4.57-4.47 (m, 1H), 3.73 (s, 3H), 3.56 (s, 1H), 3.34-3.27 (m, 1H), 3.26-3.02 (m, 4H), 2.12 (td, J=14.3, 4.4 Hz, 1H), 1.95 (dd, J=23.3, 15.7 Hz, 3H), 1.83 (s, 3H). ESI (M+H)+=540.
By using 5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-formic acid and (4S,5S)-2-(2-methylsulfonamidoethyl)-4-(3,4-difluorophenyl)-5-amidopiperidine-1-tert-butyl formate as raw materials, Compound 127 is prepared and obtained according to the methods as in Example 144, the yield is 33%; 1H NMR (400 MHz, MeOD) δ 7.44 (s, 1H), 7.24-7.15 (m, 2H), 7.14-7.03 (m, 2H), 4.45 (td, J=11.4, 4.8 Hz, 1H), 3.79 (t, J=12.2 Hz, 1H), 3.65 (s, 3H), 3.46-3.24 (m, 3H), 3.21-3.12 (m, 2H), 2.90 (s, 3H), 2.24-1.98 (m, 4H). ESI (M+H)+=476.
By using 5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-formic acid and (4S,5S)-2-(2,3-dihydroxypropyl)-4-(3,4-difluorophenyl)-5-nitropiperidine-1-tert-butyl formate as raw materials, Compound 128 is prepared and obtained according to the methods as in Example 144, the yield is 47%; 1H NMR (500 MHz, MeOD) δ 7.56 (s, 1H), 7.31 (s, 1H), 7.30-7.25 (m, 1H), 7.25-7.19 (m, 1H), 7.17 (s, 1H), 4.57 (td, J=11.3, 5.1 Hz, 1H), 4.01 (d, J=2.5 Hz, 1H), 3.93-3.88 (m, 1H), 3.77 (s, 3H), 3.68-3.59 (m, 2H), 3.46-3.36 (m, 3H), 2.37-2.31 (m, 1H), 2.27-2.16 (m, 2H), 2.02-1.96 (m, 1H). ESI (M+H)+=529.
By using 5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-formic acid and (4S,5S)-2-(2-hydroxyethyl)-4-(3,4-difluorophenyl)-5-amidopiperidine-1-tert-butyl formate as raw materials, Compound 129 is prepared and obtained according to the methods as in Example 144, the yield is 23%; 1H NMR (400 MHz, MeOD) δ 7.55 (s, 1H), 7.33-7.08 (m, 4H), 4.27 (td, J=10.5, 4.9 Hz, 1H), 3.84-3.71 (m, 5H), 3.28 (d, J=4.9 Hz, 1H), 3.15 (td, J=11.8, 3.6 Hz, 1H), 3.04-2.87 (m, 2H), 2.16-2.07 (m, 1H), 2.01-1.93 (m, 1H), 1.93-1.77 (m, 2H). ESI (M+H)+=499.
By using 5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-formic acid and (4S,5S)-2-propyl-4-(3,4-dichlorophenyl)-5-amidopiperidine-1-tert-butyl formate as raw materials, Compound 130 is prepared and obtained according to the methods as in Example 144, the yield is 44%; 1H NMR (400 MHz, MeOD) δ 7.67-7.44 (m, 3H), 7.40-7.27 (m, 2H), 4.60-4.53 (m, 1H), 3.89-3.58 (m, 4H), 3.46-3.34 (m, 3H), 2.26-1.98 (m, 3H), 1.86 (dt, J=15.3, 6.0 Hz, 1H), 1.62-1.47 (m, 2H), 1.10 (t, J=7.3 Hz, 3H). ESI (M+H)+=531.
By using 5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-formic acid and (4S,5S)-2-(2-acetoxylethyl)-4-(3,4-difluorophenyl)-5-amidopiperidine-1-tert-butyl formate as raw materials, Compound 131 is prepared and obtained according to the methods as in Example 144, the yield is 29%; 1H NMR (400 MHz, MeOD) δ 7.55 (s, 1H), 7.37-7.08 (m, 4H), 4.44-4.11 (m, 3H), 3.76 (s, 3H), 3.28-3.10 (m, 2H), 3.00 (dd, J=12.8, 4.8 Hz, 1H), 2.95-2.83 (m, 1H), 2.17 (tt, J=13.1, 6.5 Hz, 1H), 2.03 (s, 3H), 2.02-1.91 (m, 2H), 1.90-1.82 (m, 1H). ESI (M+H)+=541.
By using 5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-formic acid and (4S,5S)-2-(2-trifluoroacetoxylethyl)-4-(3,4-difluorophenyl)-5-amidopiperidine-1-tert-butyl formate as raw materials, Compound 132 is prepared and obtained according to the methods as in Example 144, the yield is 21%; ESI (M+H)+=595.
By using 4-(4-chloro-1-methyl-1H-pyrazol-5-yl)thiophene-2-formic acid and Intermediate 7-5 as raw materials, Compound 133 is prepared and obtained according to the methods as in Example 144, the yield is 51%; ESI (M+H)+=508.
Adding Intermediate 1-5 (380 mg, 2 mmol) into 2-HEAF ion solution, slowly dropwise adding m-fluorobenzaldehyde (Intermediate 4-1, 372 mg, 3 mmol) thereto in an ice bath, and reacting overnight at room temperature. After the reaction is completed, adding water for diluting, extracting thereof with ethyl acetate 3 times, merging the layer of ethyl acetate, washing the organic layer with saturated saline solution 3 times, and drying it with anhydrous sodium sulfate. Carrying out column chromatography with ethyl acetate/petroleum ether system, recycling the solvent under reduced pressure, and purifying by column chromatography, 473 mg of light yellow solid (Intermediate 9-2) is obtained and the yield is 80%.
Dissolving Intermediate 9-2 (592 mg, 2 mmol), ((S)-(−)-α,α-diphenyl-2-pyrrylmethyl) trimethylsilyl ether (33 mg, 0.1 mmol), benzoic acid (25 mg, 0.2 mmol) in water (5 ml), slowly dropwise adding 0.1 ml of n-valeraldehyde under vigorous stirring, and then reacting overnight after the adding is completed. After the reaction is completed, extracting the water layer with ethyl acetate 3 times, merging the layer of ethyl acetate, washing the organic layer with saturated NaHCO3 solution 3 times and with saturated saline solution 3 times, drying it with anhydrous sodium sulfate, and recycling the solvent under reduced pressure, yellow oily matter is obtained (Intermediate 9-3).
Dissolving the oily matter obtained from the previous step (Intermediate 9-3) in anhydrous dichloromethane (5 ml), sequentially slowly dropwise adding triethyl silicane (700 mg, 6 mmol) and aether boron trifluoride (426 mg, 3 mmol) thereto in an ice bath. After the reaction is completed, slowly adding saturated NaHCO3 solution (10 ml), extracting thereof with dichloromethane 3 times, merging the organic layer, washing the organic layer with saturated saline solution 3 times, and drying it with anhydrous sodium sulfate. Recycling the solvent under reduced pressure, primary product is obtained (Intermediate 9-4) and input to the next reaction.
Dissolving above primary product (Intermediate 9-4) in ethyl acetate (10 ml), adding 50 mg of 10% Pd/C thereto, hydrogenating overnight at room temperature, suction filtrating after the reaction is completed, and spin drying the filtrate, 290 mg of oily matter (Intermediate 9-5) is obtained, the yield of three steps is 43%; ESI (M+H)+=337.
Dissolving Intermediate 8-2 (95 mg, 0.345 mmol), 1-hydroxybenzotriazole (HOBt) (78.62 mg, 0.517 mmol) and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride EDC.HCl (98.8 mg, 0.517 mmol) in anhydrous dichloromethane (4 ml), after stirring for 10 min in an ice bath, adding diisopropylethylamine (0.115 ml, 1.21 mmol), after continue stirring for 15 min in an ice bath, slowly adding dichloromethane solution (4 ml) dissolved with Intermediate 9-5 (118 mg, 0.35 mmol), and stirring overnight at room temperature. After the reaction is completed, pouring the reaction liquid to 15 ml of water, extracting thereof with dichloromethane 3 times, merging the organic phase, washing with saturated sodium chloride twice, drying it with anhydrous sodium sulfate, and spin drying; dissolving the obtained residue to a small amount of acetate ethyl, slowly adding HCl saturated ethyl acetate in an ice bath, spin drying after reacting for 3 h at room temperature, adding saturated NaHCO3 solution thereto, extracting the reaction liquid with ethyl acetate 2 times, merging the organic phase, drying it with anhydrous sodium sulfate, and purifying by column chromatography on silica gel, 100 mg of white powder (Compound 134) is obtained and the yield is 59%; 1H NMR (500 MHz, MeOD) δ 7.95 (dd, J=8.3, 1.2 Hz, 1H), 7.75 (d, J=1.2 Hz, 1H), 7.41-6.93 (m, 4H), 4.55 (td, J=11.7, 4.4 Hz, 1H), 3.82 (d, J=6.1 Hz, 3H), 3.68-3.55 (m, 2H), 3.22-3.07 (m, 1H), 2.98-2.81 (m, 2H), 2.31-2.13 (m, 1H), 2.08-2.00 (m, 1H), 1.46-1.15 (m, 4H), 0.80 (dd, J=16.0, 9.1 Hz, 3H). ESI (M+H)+=495.
By using 5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)thiophene-2-formic acid and (3S, 4S,5R)-4-(3-fluorophenyl)-3-amido-5-methylpiperidin-1-tert-butyl formate as raw materials, Compound 135 is prepared and obtained according to the methods as in Example 174, and the yield is 61%; 1H NMR (500 MHz, MeOD) δ 7.95 (dd, J=8.3, 1.1 Hz, 1H), 7.71 (dd, J=28.5, 1.1 Hz, 1H), 7.37-7.21 (m, 2H), 7.18-6.96 (m, 2H), 4.57 (td, J=11.5, 4.6 Hz, 1H), 3.81 (d, J=6.3 Hz, 3H), 3.69-3.59 (m, 1H), 3.51 (dt, J=23.5, 10.0 Hz, 1H), 3.20-3.07 (m, 1H), 2.92 (t, J=12.5 Hz, 1H), 2.78 (dt, J=28.3, 11.3 Hz, 1H), 2.38-2.20 (m, 1H), 2.10-1.96 (m, 1H), 0.91-0.77 (m, 3H). ESI (M+H)+=467.
By using 5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-formic acid and (3S, 4S, 5R)-4-(3-fluorophenyl)-3-amido-5-methylpiperidin-1-tert-butyl formate as raw materials, Compound 136 is prepared and obtained according to the methods as in Example 174, and the yield is 66%; 1H NMR (500 MHz, MeOD) δ 7.55 (d, J=1.4 Hz, 1H), 7.40-7.20 (m, 3H), 7.17-6.97 (m, 2H), 4.70-4.59 (m, 1H), 3.75 (d, J=4.0 Hz, 3H), 3.60 (dd, J=12.1, 4.1 Hz, 1H), 3.51 (dt, J=9.9, 4.5 Hz, 1H), 3.12 (td, J=12.1, 5.1 Hz, 1H), 2.91 (t, J=12.5 Hz, 1H), 2.85-2.70 (m, 1H), 2.35-2.21 (m, 1H), 2.04 (d, J=4.4 Hz, 1H), 0.83 (dd, J=9.5, 6.7 Hz, 3H). ESI (M+H)+=451.
By using 5-chloro-4-(4-chloro-1-methyl-1H-pyrazol-5-yl)furan-2-formic acid and Intermediate 9-5 as raw materials, Compound 137 is prepared and obtained according to the methods as in Example 174, and the yield is 60%; 1H NMR (500 MHz, MeOD) δ 7.56 (s, 1H), 7.40-7.29 (m, 1H), 7.24 (s, 1H), 7.19-6.97 (m, 3H), 4.68-4.55 (m, 1H), 3.75 (d, J=4.0 Hz, 3H), 3.69-3.54 (m, 2H), 3.19-3.05 (m, 1H), 2.99-2.81 (m, 2H), 2.28-2.14 (m, 1H), 2.08-1.98 (m, 1H), 1.44-1.15 (m, 4H), 0.79 (dd, J=16.0, 9.0 Hz, 3H). ESI (M+H)+=479.
By using 5-(4-chloro-1-methyl-1H-pyrazol-5-yl)thiophene-2-formic acid and (3S,4S)-3-amido-4-(3-fluorophenyl)piperidin-1-tert-butyl formate as raw materials, Compound 138 is prepared and obtained according to the methods as in Example 174, and the yield is 53%; 1H NMR (500 MHz, MeOD) δ 7.65 (d, J=3.8 Hz, 1H), 7.55 (s, 1H), 7.37-7.30 (m, 2H), 7.18 (d, J=7.8 Hz, 1H), 7.13 (d, J=10.1 Hz, 1H), 6.98 (m, 1H), 4.60 (m, 1H), 3.89 (s, 3H), 3.67-3.61 (m, 1H), 3.56 (d, J=12.7 Hz, 1H), 3.22 (t, J=13.0 Hz, 2H), 3.15 (t, J=12.0 Hz, 1H), 2.23 (d, J=12.4 Hz, 1H), 2.14-2.04 (m, 1H). ESI (M+H)+=419.
By using 4-chloro-5-(4-chloro-1-methyl-1H-pyrazol-5-yl)thiophene-2-formic acid and (3S, 4S)-3-amido-4-(3-fluorophenyl)piperidin-1-tert-butyl formate as raw materials, Compound 139 is prepared and obtained according to the methods as in Example 174, and the yield is 52%; ESI (M+H)+=453.
By using 5-(1-methyl-4-(pyridinyl-4-yl)-1H-pyrazol-5-yl)thiophene-2-formic acid and (3S, 4S)-3-amido-4-(3-fluorophenyl)piperidin-1-tert-butyl formate as raw materials, Compound 140 is prepared and obtained according to the methods as in Example 174, and the yield is 34%; 1H NMR (500 MHz, MeOD) δ 8.61 (d, J=6.7 Hz, 2H), 8.29 (s, 1H), 7.85 (d, J=6.7 Hz, 2H), 7.80 (d, J=3.8 Hz, 1H), 7.38-7.33 (m, 2H), 7.22 (d, J=7.7 Hz, 1H), 7.15 (d, J=9.9 Hz, 1H), 7.00 (td, J=8.4, 2.1 Hz, 1H), 4.61 (dt, J=11.6, 5.8 Hz, 1H), 3.83 (s, 3H), 3.63-3.59 (m, 1H), 3.58-3.53 (m, 1H), 3.31-3.12 (m, 3H), 2.22 (d, J=12.4 Hz, 1H), 2.13-2.06 (m, 1H). ESI (M+H)+=462.
By using 5-(1-methyl-4-hydroxylmethyl-1H-pyrazol-5-yl)thiophene-2-formic acid and (3S, 4S)-3-amido-4-(3-fluorophenyl)piperidin-1-tert-butyl formate as raw materials, Compound 141 is prepared and obtained according to the methods as in Example 174, and the yield is 39%; 1H NMR (500 MHz, MeOD) δ 7.80 (s, 1H), 7.69 (d, J=3.9 Hz, 1H), 7.37-7.30 (m, 2H), 7.20 (d, J=7.7 Hz, 1H), 7.13 (d, J=10.0 Hz, 1H), 6.98 (dd, J=11.8, 5.1 Hz, 1H), 4.61 (td, J=11.6, 4.3 Hz, 1H), 4.29 (s, 2H), 3.94 (s, 3H), 3.66-3.62 (m, 1H), 3.56 (d, J=12.6 Hz, 1H), 3.28-3.12 (m, 3H), 2.22 (d, J=12.9 Hz, 1H), 2.14-2.06 (m, 1H). ESI (M+H)+=415.
By using 5-(1-methyl-4-methoxycarbonyl-1H-pyrazol-5-yl)thiophene-2-ethyl formate and (3S, 4S)-3-amido-4-(3-fluorophenyl)piperidin-1-tert-butyl formate as raw materials, Compound 142 is prepared and obtained by amide condensation, hydrolysis, deprotection steps and the like according to the methods as in Example 174, the yield is 54%; 1H NMR (500 MHz, MeOD) δ 7.96 (s, 1H), 7.63 (d, J=3.1 Hz, 1H), 7.39-7.30 (m, 1H), 7.24 (d, J=3.2 Hz, 1H), 7.21-7.09 (m, 2H), 6.98 (t, J=7.6 Hz, 1H), 4.62 (s, 1H), 3.77 (s, 3H), 3.63 (d, J=7.0 Hz, 1H), 3.54 (t, J=10.0 Hz, 1H), 3.28-3.09 (m, 3H), 2.22 (d, J=14.0 Hz, 1H), 2.08 (d, J=11.8 Hz, 1H). ESI (M+H)+=429.
By using 5-(1-methyl-4-(1-hydroxyethyl)-1H-pyrazol-5-yl)thiophene-2-formic acid and (3S, 4S)-3-amido-4-(3-fluorophenyl)piperidin-1-tert-butyl formate as raw materials, Compound 143 is prepared and obtained according to the methods as in Example 174, and the yield is 34%; ESI (M+H)+=429.
By using 5-(1-methyl-4-vinyl-1H-pyrazol-5-yl)thiophene-2-formic acid and (3S,4S)-3-amido-4-(3-fluorophenyl)piperidin-1-tert-butyl formate as raw materials, Compound 144 is prepared and obtained according to the methods as in Example 174, and the yield is 64%; ESI (M+H)+=411.
By using 5-(1-methyl-4-ethyl-1H-pyrazol-5-yl)thiophene-2-formic acid and (3S, 4S)-3-amido-4-(3-fluorophenyl)piperidin-1-tert-butyl formate as raw materials, Compound 145 is prepared and obtained according to the methods as in Example 174, and the yield is 62%; 1H NMR (500 MHz, MeOD) δ 7.92 (s, 1H), 7.76 (d, J=3.8 Hz, 1H), 7.36-7.29 (m, 2H), 7.21 (d, J=7.7 Hz, 1H), 7.15 (d, J=10.0 Hz, 1H), 6.96 (td, J=8.5, 2.2 Hz, 1H), 4.64 (td, J=11.6, 4.3 Hz, 1H), 3.93 (s, 3H), 3.66-3.62 (m, 1H), 3.57 (d, J=12.6 Hz, 1H), 3.32-3.17 (m, 3H), 2.57-2.50 (m, 2H), 2.25-2.07 (m, 2H), 1.21-1.16 (m, 3H). ESI (M+H)+=413.
By using 5-(1-methyl-1H-pyrazol-5-yl)thiophene-2-formic acid and (3S, 4S)-3-amido-4-(3-fluorophenyl)piperidin-1-tert-butyl formate as raw materials, Compound 146 is prepared and obtained according to the methods as in Example 174, and the yield is 54%; 1H NMR (500 MHz, MeOD) δ 7.98-7.96 (m, 1H), 7.70 (d, J=4.0 Hz, 1H), 7.42 (d, J=3.9 Hz, 1H), 7.24 (dd, J=14.0, 7.9 Hz, 1H), 7.15 (d, J=7.8 Hz, 1H), 7.09 (dd, J=10.0, 1.9 Hz, 1H), 6.88 (td, J=8.5, 2.3 Hz, 1H), 6.78 (d, J=2.6 Hz, 1H), 4.60 (td, J=11.6, 4.3 Hz, 1H), 4.05 (s, 3H), 3.60-3.57 (m, 1H), 3.51 (d, J=12.7 Hz, 1H), 3.25-3.13 (m, 3H), 2.17-2.04 (m, 2H). ESI (M+H)+=385.
By using 5-(1-methyl-4-bromo-1H-pyrazol-5-yl)thiophene-2-formic acid and (3S,4S)-3-amido-4-(3-fluorophenyl)piperidin-1-tert-butyl formate as raw materials, Compound 147 is prepared and obtained according to the methods as in Example 174, and the yield is 68%; 1H NMR (500 MHz, MeOD) δ 7.68 (d, J=3.9 Hz, 1H), 7.56 (s, 1H), 7.35-7.30 (m, 1H), 7.28 (d, J=3.9 Hz, 1H), 7.20 (d, J=7.7 Hz, 1H), 7.14 (d, J=9.9 Hz, 1H), 6.96 (td, J=8.5, 2.2 Hz, 1H), 4.63 (td, J=11.5, 4.1 Hz, 1H), 3.87 (s, 3H), 3.67-3.61 (m, 1H), 3.57 (d, J=12.3 Hz, 1H), 3.30-3.14 (m, 3H), 2.22 (d, J=13.7 Hz, 1H), 2.17-2.06 (m, 1H). ESI (M+H)+=463.
By using 5-(1-methyl-4-phenyl-1H-pyrazol-5-yl)thiophene-2-formic acid and (3S, 4S)-3-amido-4-(3,4-difluorophenyl)piperidin-1-tert-butyl formate as raw materials, Compound 148 is prepared and obtained according to the methods as in Example 174, and the yield is 59%; 1H NMR (500 MHz, CDCl3) δ 7.78 (s, 1H), 7.55 (d, J=3.2 Hz, 1H), 7.19-6.94 (m, 9H), 6.82 (t, J=7.5 Hz, 1H), 4.47 (t, J=9.4 Hz, 1H), 3.71 (s, 3H), 3.46-3.32 (m, 2H), 3.13-2.95 (m, 3H), 2.10-1.91 (m, 2H). ESI (M+H)+=479.
By using 4-chloro-5-(4-chloro-1-methyl-1H-pyrazol-5-yl)thiophene-2-formic acid and (4S, 5S)-4-(3-fluorophenyl)-2-(2-(1-methylamino)-2-oxyethyl)-5-amidopiperidine-1-tert-butyl formate as raw materials, Compound 149 is prepared and obtained according to the methods as in Example 174, and the yield is 46%; ESI (M+H)+=524.
By using 5-(4-chloro-1-methyl-1H-pyrazol-5-yl)thiophene-2-formic acid and (3S, 4S, 5R)-4-(3-fluorophenyl)-3-amido-5-methylpiperidin-1-tert-butyl formate as raw materials, Compound 150 is prepared and obtained according to the methods as in Example 174, and the yield is 56%; 1H NMR (500 MHz, MeOD) δ 7.64-7.52 (m, 2H), 7.39-7.22 (m, 3H), 7.19-6.95 (m, 2H), 4.58 (s, 1H), 3.89 (d, J=3.9 Hz, 3H), 3.62 (d, J=7.0 Hz, 1H), 3.50 (dd, J=14.0, 7.1 Hz, 1H), 3.12 (s, 1H), 2.91 (s, 1H), 2.87-2.70 (m, 1H), 2.28 (s, 1H), 2.03 (d, J=4.5 Hz, 1H), 0.95-0.75 (m, 3H). ESI (M+H)+=433.
By using 5-(4-chloro-1-methyl-1H-pyrazol-5-yl)thiophene-2-formic acid and Intermediate 9-5 as raw materials, Compound 151 is prepared and obtained according to the methods as in Example 174, and the yield is 71%; 1H NMR (500 MHz, MeOD) δ 7.59 (d, J=3.9 Hz, 1H), 7.55 (d, J=4.2 Hz, 1H), 7.39-7.30 (m, 2H), 7.18-6.97 (m, 3H), 4.55 (td, J=11.7, 4.5 Hz, 1H), 3.89 (d, J=4.0 Hz, 3H), 3.63 (dd, J=12.5, 4.0 Hz, 2H), 3.11 (t, J=12.1 Hz, 1H), 2.99-2.81 (m, 2H), 2.21 (dd, J=7.6, 3.8 Hz, 1H), 1.49-1.06 (m, 4H), 0.80 (dd, J=15.9, 9.0 Hz, 3H). ESI (M+H)+=461.
By using 5-(4-chloro-1-methyl-1H-pyrazol-5-yl)thiophene-2-formic acid and Intermediate 7-5 as raw materials, Compound 152 is prepared and obtained according to the methods as in Example 174, and the yield is 69%; 1H NMR (500 MHz, MeOD) δ 7.71 (d, J=4.0 Hz, 1H), 7.57 (d, J=5.2 Hz, 1H), 7.35 (d, J=3.9 Hz, 1H), 7.34-7.28 (m, 1H), 7.27-7.15 (m, 2H), 4.50 (td, J=11.5, 4.9 Hz, 1H), 4.12 (dd, J=13.1, 6.0 Hz, 1H), 3.91 (s, 3H), 3.53-3.45 (m, 1H), 3.41 (dt, J=19.3, 8.2 Hz, 2H), 3.06 (dd, J=16.3, 10.0 Hz, 1H), 2.81 (d, J=5.7 Hz, 3H), 2.76 (dd, J=16.3, 5.2 Hz, 1H), 2.29-2.19 (m, 1H), 2.09 (d, J=13.2 Hz, 1H). ESI (M+H)+=508.
The compound AZD5363 (NCT02208375, NCT02208375, NCT01625286) which enters in clinical phase II studies as a positive control, the inhibitory effects in vitro (IC50) of Compounds on common tumor cell strains (Human ovarian cancer cell strain OVCAR-8 and human colon cancer cell strain HCT-116) are determined using MTT assay, meanwhile, the inhibitory activities (IC50) thereof against Akt1 enzyme are assessed using commercial Akt1 kit.
Methods and results of pharmacological experiments on antitumor activity of compounds in the present invention are as follows:
First, inhibitory activity on tumor proliferation in vitro is determined and the structure activity relationship is preliminarily studied, in which different solid tumor cell strains are selected for determining the antitumor activities in vitro of the synthesized compounds.
Experimental materials:
Cell strains: Human ovarian cancer cell strain (OVCAR8), colon adenocarcinoma cell strain (HCT-116)
Preparing method of medicines: Dissolving the medicine in DMSO to make a 50 mM stock solution, and diluting it according to a certain ratio to obtain 5 different concentrations.
Culture the tumor cell strains in vitro:
Two selected tumor cells OVCAR8 and HCT-116 are incubated at 37° C. in a 5% CO2 incubator, which are subcultured when cells grow up to a density of 70˜90% (adherent cells are subcultured after digesting by Duck's EDTA) for later experiments.
Compounds are dissolved and diluted by dimethyl sulfoxide (DMSO), and tumor cells OVCAR8 and HCT-116 are seeded in a 96-well plate at a density of 4000 cells/200 μl/well, 1 μl of the compounds are added to each well with the final concentration of 50 μM, 10 μM, 2 μM, 0.4 μM and 0.08 μM, which is incubated for 72 h at 37° C. in a 5% CO2 incubator with DMSO (1%) as blank control. After incubating for 72 h, MTT with a final concentration of 0.25 mg/mL is added, which is maintained for 4 h at 37° C. in a 5% CO2 incubator, and then the medium is suck dried, 100 μl of DMSO is added to each well, the absorbance (OD value) is determined at 570 nm by enzyme-linked immunometric meter, thus obtained data is used to calculate IC50.
The calculating formula of cell inhibitory rate is: cell inhibitory rate %=(the OD value of the control group−the OD value of the medication group)/the OD value of the control group×100%, the half maximal inhibitory concentration (IC50) is obtained by Bliss method.
Second, method for determining the inhibitory activities of substituted nitrogen-containing heterocyclic derivatives in the present invention against Akt1 enzyme:
The inhibitory activities of compounds against AKT1/PKBα are determined by using AKT1/PKBα KinEASE™ FP Fluorescein Green Assay (Green fluorescence detection system of Kinase).
Principle adopted by the fluorescence polarization detection of protein kinase B is a competitive reaction: the phosphorylated tracers labeled with fluorescence will compete with unlabeled phosphorylated products produced by reacting with protein kinase B to combine anti-serine antibodies. In a reaction mixture without phosphorylated products, the combination a part of fluorescent tracers and antibodies will lead to a higher polarization value. However, in the reaction mixture containing phosphorylated products, fewer tracers will combine with antibodies (fluorescent tracers are replaced from the antibodies), and signals sent our occur depolarizing. Therefore, the change of polarization is directly related to the activity of protein kinase B in the reaction.
Compounds in the present invention and the positive control AZD5363 are dissolved with dimethyl sulfoxide (DMSO), and are diluted to a concentration of 50 μM. Each of 0.25 μl of compounds with a concentration of 50 μM and the positive control are added to a 384-well plate at room temperature, and each sample is provided with three parallel wells, and then 10 μl of STK Substrate 3 Working Solution, 5 μl of AKT1/PKBα Working Solution, 10 μl of ATP Working Solution are added to each sample respectively, the mixtures are slightly vibrated and shaked for a few minutes. Reaction is carried out just after the adding 10 μl of ATP Working Solution, from this time, reacting for 1 h. After 1 h, 5 μl of STK Stop Mix and 5 μl of STK Antibody Mix are added to each sample respectively to stop the reaction. The samples are maintained for 4 h at room temperature after adding is completed, the polarization values of samples are determined by fluorescence polarization of Microplate Reader (Detection of the signals is valid within 24 h), the inhibitory rate of compounds against the enzyme is calculated by polarization value, and thereby IC50 is calculated.
Experiment is set up four control groups at the same time, which are Buffer Control Wells, Tracer Control Wells, No Enzyme Wells and blank control of dimethyl sulfoxide, respectively. Thus obtained data are used to calculate the inhibitory rate (the preparation methods of above various solution required for determining the activities of compounds against enzyme refer to the specification of the kit of AKT1/PKBα KinEASE™ FP Fluorescein Green Assay Catalog #32-021).
It can be seen from the activity data in above table, all the tested compounds show significant inhibitory activities against Akt1 kinase, IC50 values of most compounds are less than 0.1 μM; IC50 values of a part of compounds are less than 0.009 μM, better than or equal to the positive compound AZD5363 (IC50=0.009 μM, the compound is a potent Akt1 inhibitor, which is in clinical phase II study for treating human breast cancer, NCT01625286); IC50 values of part of compounds can reach to a pmol level, such as: Compound 115 (0.0008 μM), Compound 123 (0.0001 μM), Compound 127 (0.0003 μM) and Compound 134 (0.0003 μM) and the like, which is significantly better than the positive control AZD5363 (0.009 μM). Therefore, compounds in the present invention can be used as a kind of Akt inhibitors with novel structure.
In addition, the majority of tested compounds show potent anti-proliferative activities against both of two tumor cell strains (both of the IC50 values are less than 10 μM), IC50 values of most compounds to OVCAR-8, HCT-116 tumor cell strains are less than the positive drug AZD5363 (7.27 μM, 5.20 μM), which is better than or equal to the positive compound AZD5363; IC50 values of part of compounds can reach to less than 0.1 μM, such as Compound 115 (0.78 μM, 0.56 μM), Compound 123 (0.09 μM, 0.07 μM), Compound 134 (0.53 μM, 0.69 μM), which is significantly better than the positive compound AZD5363. Therefore, compounds involved in the present invention have potent antitumor activities.
In conclusion, substituted nitrogen-containing heterocyclic derivatives involved in the present invention can be used as Akt inhibitors, having a broad applicating prospect for cancer treatment.
| Number | Date | Country | Kind |
|---|---|---|---|
| 201410108818.5 | Mar 2014 | CN | national |
| 201410124591.3 | Mar 2014 | CN | national |
| 201510101220.8 | Mar 2015 | CN | national |
| Filing Document | Filing Date | Country | Kind |
|---|---|---|---|
| PCT/CN2015/704813 | 3/21/2015 | WO | 00 |
